The definite existence of any virus, including a retrovirus, can be
proven only by isolating it. For nearly half a century retroviruses have
been isolated by banding in density gradients. It is accepted that the
procedures incorporated into this method, which is by no means perfect,
have not been followed by the researchers who claim isolation of the
human immunodeficiency virus, HIV-1. Nonetheless, it is said that at
present, there is ample evidence that HIV has been isolated and shown to
be a unique exogenous retrovirus.(1) In this critique we have analysed
the relevant data that purport to prove that HIV has been isolated. To
simplfy the presentation for readers of this article, the major
arguments (1) for HIV isolation are used as the headings in the
discussion. Since the topic is both complex and controversial it is
necessary to present substantial original data and sometimes to repeat
it in order to critically assess the basis for the view that HIV has
been isolated.

(Please note that some Greek characters may not
print. Lamda may appear as the control character (^) and alpha, beta and
gamma be g, b or a.)

1. "In 1983 Montagnier et al isolated a retrovirus".

In the 1983 Montagnier et al study there is no proof of virus
isolation by "the most rigorous method available to date". Nor did they
follow the "traditional...Pasteur rules". How then did they isolate a
retrovirus? Even if Montagnier and his colleagues or others had followed
the "Pasteur rules", since "viral and cellular proteins, and cellular
contaminants...copurify with virus purified by conventional density
gradients",(1) there is no reason to accept any claim of HIV isolation
by any research group who did not use "the most rigorous method
available to date, i.e. molecular cloning of infectious HIV DNA".
However, to prove that HIV "has been isolated" by "the most rigorous
method available to date", virus cloning, one must start with HIV RNA
(DNA). Since the propriety of naming an RNA "HIV RNA" is contingent upon
prior isolation of a particle proven to be a retrovirus, on this basis
alone, "the most rigorous method available to date, i.e. molecular
cloning of infectious HIV DNA", cannot prove HIV isolation.

2. "reverse transcriptase associated with such particles".

There is not one single study which proves that the enzyme present in
the "growth medium" or even in the material which in sucrose density
gradients bands at 1.16 gm/ml, (the density which defines retroviral
particles), and which catalyses the transcription of RNA into DNA, is a
constituent of particles of any kind, much less of retroviral-like
particles or a unique retrovirus. The only association between
"particles" and "reverse transcriptase" (RT) arises from experiments
which show that some cultures/cocultures with tissues from AIDS patients
exhibit both particles, many of which are not even retroviral-like, and
transcription of the synthetic RNA template-primer A(n).dT15. However,
this does not constitute proof of the existence of RT or RT as a
constituent of a retroviral particle. Furthermore, since: (a) the
presence of reverse transcriptase (RT) is proven indirectly, that is, by
demonstrating transcription of the RNA template-primer A(n).dT15; (b)
the template-primer A(n).dT15 can be transcribed not only by RT but by
other cellular DNA polymerases. All the cellular DNA polymerases, à, á
and y, can copy A(n).dT15 (2). In fact, in 1975, an International
Conference on Eukaryotic DNA polymerases, which included Baltimore and
Gallo (3) defined DNA polymerase y, "a component of normal cells" (4),
"found to be widespread in occurrence" (2), whose activity can be
increased by many factors including PHA stimulation (5), as the enzyme
which "copies A(n).dT15 with high efficiency but does not copy DNA
well";(3) it is impossible to say that the polymerase in the "growth
medium" or in the material banding at 1.16 gm/ml which catalyses reverse
transcription of A(n).dT15 is RT or one of a number of other cellular
DNA polymerases.

3. "...indeed, each of these criteria could reflect another
retrovirus, and some of these criteria, eg, particles and proteins,
could reflect non-viral material altogether".

Although the HIV/AIDS experts, including Montagnier, Gallo and
Barr-Sinoussi claim that RT is "unique to retroviruses" and "the
hallmark of a retrovirus",(6-8) this is not the case, a fact accepted by
some of the best known scientists.(9) "Reverse transcriptase (RT) was
first discovered as an essential catalyst in the biological cycle of
retroviruses. However, in the past years, evidence has accumulated
showing that RTs are involved in a surprisingly large number of
RNA-mediated transcriptional events that include both viral and nonviral
genetic entities...the possibility that reverse transcription first took
place in the early Archean" is supported by a number of facts and "the
hypothesis that RNA preceded DNA as cellular genetic material". (10)
According to Varmus: "Reverse transcription was assigned a central role
in the replication of other viruses [hepatitis B and cauliflower mosaic
viruses] and in the transposition and generation of other kinds of
eukaryotic DNA". (11) "The hepatitis B viruses (HBVs) are small DNA
viruses that produce persistent hepatic infections in a variety of
animal hosts and replicate their DNA genomes via reverse transcription
of an RNA intermediate. All members of this family contain an open
reading frame (ORF), "P" (for pol), which is homologous to retroviral
pol genes" (pol=polymerase).(12) "Hepatitis B virus (HBV) resembles
retroviruses, including HIV, in several respects. In particular, both
viruses contain reverse transcriptase, and replicate through an RNA
intermediate". Because of this, it has been suggested that hepatitis B
infection should be treated with the same antiretroviral agents as HIV
infection.(13). At present, evidence exists which shows that although
the major target organ for hepatitis B virus is the liver, cells other
than hepatocytes "including peripheral blood lymphocytes and monocytes,
may become infected with HBV" (14). Lymphocyte stimulation in general
and PHA stimulation in particular is associated with production of
hepatitis B virus from peripheral blood lymphocytes in patients infected
with HBV including "viral replication in chronic hepatitis B infection
of childhood".(15,16) According to Doolittle et al, "...there are many
reverse transcriptase-bearing entities other than retroviruses,
including mobile elements found in a wide variety of eukaryocytes, some
plant and animal DNA viruses, and even some introns" (17). In one of his
most recent publications, one of the best known retrovirologists, Robin
Weiss from the Institute of Cancer Research, London, UK, wrote, "Now we
know that a broader group of genetic elements than retroviruses utilise
reverse transcription at some stage of replication; these include
hepadnaviruses (including hepatitis B virus), cauliflower mosaic virus
and retrotransposons of eukaryotes and prokaryotes. Indeed lamivudine
may find a place in the treatment of hepatitis B infections as well as
HIV".(18) In other words, RT does not seem to be more specific to
retroviruses than ATPase, an enzyme now known to be ubiquitous but
which, before the discovery of RT, was used to both detect and quantify
retroviruses.(19) Since in all the HIV literature, by HIV isolation is
meant nothing more than the detection of "HIV particles", proteins and
RT (and frequently only one of them), and since any or all of these
phenomena "could reflect non-viral material altogether", does it not
therefore follow that HIV could reflect non-viral material
altogether?

4. "HIV antigens or proteins associated with such
particles".

To date nobody has presented evidence that the "HIV antigens or
proteins" are constituents of retrovirus particle or even a
retrovirus-like particle let alone a unique retrovirus, HIV.

5.1 In the 1983 paper entitled "Isolation of a T-lymphotropic
retrovirus from a patient at risk for acquired immune deficiency
syndrome (AIDS)",(20) where Montagnier and his colleagues reported the
"isolation" of their "HIV" strain, cells obtained from a lymph node
biopsy of a gay man with lymphadenopathy (lymphadenopathy syndrome
[LAS]) were put in culture with PHA, IL-2 and antiserum to human
interferon. (The latter had previously been shown in mice to lead to
"increased retrovirus production by a factor of 10 to 50"). After 15
days RT activity was detected using the synthetic primer- template
A(n).dT15. The reverse transcription of A(n)dT15 was considered proof
that a retrovirus was present in the lymph node cells. The finding of
the same activity in the supernatant of a co- culture of the same cells
with lymphocytes from a healthy individual was considered proof that the
retrovirus could be transmitted. In another experiment, polybrene and
supernatant from the co-cultures were added to two, three day old
umbilical cord lymphocytes cultures. After seven days "a relatively high
titer" of A(n.)dT15 transcription was detected. This was considered
proof not only of transmission but isolation as well. "That this new
isolate was a retrovirus was further indicated by its density in a
sucrose density gradient, which was 1.16, and by its labelling with [3H]
uridine (fig. 1)". In figure 1 evidence was presented that A(n)dT12- 18
could be transcribed by the material from the supernatant of the
umbilical cell cultures which, in sucrose density gradients, banded at
1.16 gm/ml. The "infected" umbilical cord lymphocytes as well as "HTLV-
producing" cells were lysed. The proteins from a "cell extract" obtained
from the lysates were reacted with the sera from the patient with
lymphadenopathy, another patient with "multiple adenopathies", a healthy
individual, a normal goat and goat antiserum "to HTLV-I p24". Many
proteins from both cell types but especially from the "infected"
umbilical cords, reacted with ALL sera. However, the "infected"
umbilical cord cells did not react with the antiserum to "HTLV-I p24".
The proteins from the culture supernatant which banded at 1.16 gm/ml
were also reacted with the sera but instead of the goat anti-p25
antiserum they used sera from another healthy donor. In the published
strips it is difficult if not impossible to distinguish any reactive
bands with any serum. In the text it is stated "three major proteins
could be seen: the p25 protein and proteins with molecular weights of
80.000 and 45.000" in the strip with the serum from the patient with
LAS. Montagnier et al also reported that "Electron microscopy of the
infected umbilical cord lymphocytes showed characteristic immature
particles with dense crescent (C-type) budding at the plasma membrane".
They gave no electron microscopic (EM) data on the material banding at
1.16 gm/ml but concluded "A retrovirus belonging to the family of
recently discovered human T-cell leukemia viruses (HTLV) but clearly
distinct from each previous isolate, has been isolated from a Caucasian
patient with signs and symptoms that often precede the acquired immune
deficiency syndrome (AIDS). This virus is a typical type-C RNA tumor
virus, buds from the cell membrane, prefers magnesium for reverse
transcriptase activity, and has an internal antigen (p25) similar to
HTLV p24" (20), (When it was realised that individuals who have
antibodies which react with this "virus strain" did not rapidly progress
to AIDS, without proof, the taxonomically distinct "typical type-C "
retrovirus became a taxonomically distinct, typical Lentivirus).

5.2 THE WORD "ISOLATION" IS DERIVED FROM THE LATIN
"INSULATUS" MEANING "MADE INTO AN ISLAND". IT REFERS TO THE ACT OF
SEPARATING AN OBJECT FROM ALL EXTRANEOUS MATTER THAT IS NOT THAT OBJECT.
The object of interest is not a protein, nor a fragment of RNA (DNA) but
a unique exogenous retrovirus, HIV. Nothing more and nothing less. No
such evidence was presented by Montagnier et al. Obviously, at the very
best, the finding of phenomena such as virus-like particles in cell
cultures, antibody/antigen reactions and evidence for reverse
transcription of A(n).dT15 can be considered proof only for detection of
a retrovirus, and then if and only if each are shown to be specific to
the retrovirus. This cannot be done unless the retrovirus is first
isolated. Thus it comes as no surprise that Popovic, Gallo and their
colleagues did not consider Montagnier et al's data as proof of "true
isolation".(21) [In their 1984 papers Gallo and his colleagues defined
isolation as detection of "more than one of the following:", "repeated
detection of a Mg2+ -dependent reverse transcriptase activity in
supernatant fluids; virus observed by electron microscopy (EM);
intracellular expression of virus-related antigens detected with
antibodies from seropositive donors or with rabbit antiserum to
HTLV-III; or transmission of particles". (By transmission of particles
was meant detection of RT or particles in cultures of human umbilical
cord blood, bone marrow or peripheral blood T lymphocytes, cultured with
supernatants from the "infected" cultures). Since this is no different
from the experiments that Montagnier and his colleagues performed, it
follows that Gallo and his colleagues did not prove "true isolation"
either. In fact, Gallo et al's definition of isolation raises additional
questions including: How was it possible to obtain rabbit antiserum "to
HTLV-III" before the virus was isolated and how was it possible, before
the virus was isolated, to ascertain that both the rabbit antiserum and
the patient sera used to test material from the cultures interacted
specifically with the virus? According to their definition, one can
isolate HIV even if no RT is detected. How is this possible since RT is
the "hallmark" of HIV?(22).] It is also significant that in his and his
colleagues' 1986 patent application "Improvements relating to viral
isolates and their use", Robin Weiss referred to Montagnier's "HIV
strain" as "the material". "A so-called Aids virus isolate was first
reported in 1983 by Montagnier and his colleagues in France who named
the material "Lymphadenopathy Associated Virus One"".(23) Furthermore,
isolation of a retrovirus from the umbilical cord cultures is not proof
that the retrovirus was introduced from the outside, that is, that it
originated from the patient with lymphadenopathy. All cells contain
endogenous retroviruses (see 6.3.2). In fact sperm, ova, placenta,
foetal and embryonic tissues, and to a lesser extent, umbilical cord
lymphocytes, were extensively studied because retroviruses were said to
be transmitted vertically (in the germ cell line) and because they were
thought to play a significant role in differentiation. By the beginning
of the AIDS era one or more of the following phenomena were reported
from experiments with such cells: retrovirus-like particles, reverse
transcriptase activity and retroviral antigens.(24-26) Thus such
findings cannot be proof for the existence of HIV.

Neither is the presence of antibodies in the AIDS patients, but not
in the healthy controls, which react with the proteins which band at
1.16 gm/ml, proof that such individuals are infected with an exogenous
retrovirus, HIV. For example, in a study published this year, one of the
best known retrovirologists, Reinhard Kurth, from the Paul-Ehrlich
Institute in Germany, and his colleagues, reported that 70% of
"HIV-positive patients", compared to only 3% of blood donors, had
antibodies which reacted with the retrovirus HTDV/HERV- K. However,
HTDV/HERV-K is not a retrovirus which is present only in AIDS patients,
that is, an exogenous retrovirus as HIV is said to be, but HTDV/HERV-K
is an endogenous retrovirus or, as Kurth put it, a retrovirus present
"in all of us". How is it possible then to say, based just on an
antibody test, that "Montagnier's strain", if one assumes Montagnier did
isolate such a virus, is not another endogenous retrovirus generated by
the conditions present in these patients? (see 6.3.2).

5.3 Apparently Montagnier's group found reactions between
patient sera and three proteins, p25 (p24), p45 (p41) and p80 in banded
material but only p24 was considered to be HIV protein. However, in
1984, Gallo's group reported that "No antigen from the uninfected clones
reacted with the sera, with the exception of a protein with a molecular
weight of 80.000 in H17 which bound antibodies from all of the human
serum samples tested [including normal serum] but not from rabbit or
goat serum". Because of this the p80 protein was considered to be
non-specific. "Antigens newly expressed [reactive with sera in the cell
extracts] after viral infection and recognized by the human serum used
for this analysis included p65, p55, p41, p39, p32 and p24. A large
protein with a molecular weight of approximately 130,000 and a protein
of 48,000 were also detected". Unlike Montagnier, Gallo's group also
reported that, "With normal human serum, none of the antigens was
detected (not shown)", and concluded, "These results show clearly that
the antigens detected after virus infection are either virus-coded
proteins or cellular antigens specifically induced by the
infection".(27) Gallo and his colleagues also reported that of the
proteins from the supernatant of the "infected" cultures which in
sucrose density gradients banded at 1.16 gm/ml, only two proteins, p41
and p24, reacted with patient sera and concluded that "these molecules
are the major components of the virus preparation. p24 and p41 may
therefore be considered the viral structural proteins". In the two years
following their discovery of HIV, although Montagnier's group apparently
made repeated attempts, unlike Gallo's group, they could not detect
"high molecular weight" protein which reacted with different sera but
which "was not present in the supernatant of uninfected control cells".
In experiments reported in 1985, instead of using umbilical cord
lymphocytes, they used "infected" H9 and CEM cells, two leukaemic cell
lines, and cultured (labelled) them with radioactive cysteine, 35S
cysteine, (an essential amino acid constituent of human proteins). They
reported that in the supernatant "a protein of approximately 110-120K
could be specifically immunoprecipitated by sera from pre-AIDS or AIDS
patients, in addition to core proteins, and not by sera from normal,
healthy blood donors or of laboratory workers. The protein was absent in
supernatants of uninfected T lymphocytes, T- or B- cell lines" . They
also showed that the 110K protein was a glycoprotein (gp110). For
reasons not stated, they thought that the 110K protein had a cellular
precursor. To demonstrate this, instead of using the CEM or the H9 cell
lines, they formed "A cellular hybrid, between normal T4 lymphocytes and
the MOLT-4 cell line, which was then "infected" with LAV and cultured
with radioactive cysteine. The resulting syncytia were lysed and the
proteins were reacted "with LAV-positive serum". "After 3 hr labelling,
a band of 150K was detected, Upon longer labelling, (12 hr) another band
of 135K appeared". Curiously, this was interpreted as "suggesting that
it [135] was derived from the 150K precursor" and that "either in the
cytoplasm or at the cell membrane, the gp150 is converted into the gp135
form...During virus morphogenesis, the gp135 is converted into gp110-120
by partial enzymatic removal of carbohydrates, without proteolytic
cleavage. The virus-associated [Not one single piece of their data was
derived even from a viral-like particle or material which banded at 1.16
gm/ml. All was either from "infected" cells or culture supernatant]
gp110 may itself be further processed during virus aging...besides the
main 110-120K band seen after labelling of the virus, three other thin
bands of 70K, 40K and 34K respectively, could be specially
immunoprecipitated by patients' sera. Since some of these sera did not
precipitate any gag protein, it may be assumed that these proteins are
antigenically related to gp110 and are cleavage products of the
latter".(28) This conclusion can be questioned on several grounds.
Suffice it to mention only two: (a) The culture supernatant and the
cells cannot be considered synonymous with a retrovirus. (b) Although
Montagnier et al did not comment, their data shows that many proteins,
including a p40 found in the supernatant of both "non-infected" CEM and
H9 cells react with sera from the patients with lymphadenopathy.
Somehow, without proof that they are coded by "HIV DNA", or they belong
to a retrovirus-like particle, the following proteins, gp160/150, gp
120, gp45/40, p34/32, p24, p18/17 found either in cells, supernatants,
or banding at 1.16 gm/ml in sucrose density gradients became known as
the HIV proteins. In other words, contrary to all scientific reasoning,
it was postulated that AIDS sera contain specific HIV antibodies and the
proteins with which these antibodies react were defined HIV specific
proteins.

5.4 The "HIV glycoproteins", gp160, gp120 and gp41.

(a) In 1983,(20) and again in 1984 Montagnier and his colleagues (29)
claimed that although p45/41 reacted with patient sera, this protein was
not viral but the ubiquitous cellular protein, actin. It is interesting
that even this year, the criteria used by Montagnier to define a
positive HIV Western blot is: "the presence of antibodies against
products of the env gene (gp160, gp120) and reactivity at least with one
gag gene product (WHO criteria)"(30). However to date, no other
criteria, not even the WHO criteria, exclude p41. The WHO criteria is "2
env bands (precursor, external gp, or transmembrane gp" with or without
any other bands (transmembrane=gp41)(31) Unlike Montagnier, Gallo
considers gp41 the most specific HIV protein.

In 1985, Gallo and his colleagues, comparing the fourth open reading
frame (ORF) of the "HIV DNA" which they called env-lor, with the env
genes of other retroviruses, reported that, "The predicted product of
the fourth reading frame env-lor shares many features in common with the
envelope gene precursors of other retroviruses, the most striking of
which is a hydrophobic region near the middle of the protein...The
amino-terminal domain of the translation product of the fourth open
reading frame also resembles the env protein precursors of other
retroviruses...we believe that the fourth open reading frame encodes an
env precursor...In its mature form it is probably cleaved into a large
heavily glycosylated exterior membrane protein about 481 amino acids
long and a transmembrane protein, 345 amino acids long which may be
glycosylated. The size of these predicted products agrees with the
detection of a large glycosylated protein of Mr 120-160K in HTLV-
III-infected cells which is probably the glycosylated env gene precursor
and a smaller, virion-associated gp41 which is probably the membrane
protein".(32) However, in a study published in 1987 by Gallo and his
colleagues, where they performed a "Computer- assisted analysis" of "the
amino acid sequences of the envelope protein complexes derived from the
nucleotide sequences of seven AIDS virus isolates", it was reported
that, "Although the overall sizes and structures of the seven surface
proteins are rather similar, the deduced amino acid sequences differ
substantially. On the average, only 66% of the amino acids are conserved
in the exterior part of the protein...gp41, the transmembrane part of
the envelope protein complex, shows more than 80% conserved amino
acids", but "gp41 should be about 52.000 to 54.000 daltons by
calculation".(33) Even if the molecular weight of the glycoprotein
predicted from the length of the "HIV" fourth ORF was found to be
identical to that of the protein present in the Western blot (41,000),
the claim by Gallo that the interaction of gp41 with antibodies found in
AIDS patient sera is proof that gp41 is coded by the "HIV genome", and
that both gp41 and the antibodies are specific to a retrovirus, is at
odds with what Gallo was saying in 1981. In the mid 1970s, Gallo and his
colleagues reported the isolation of the first human retrovirus, HL23V.
In fact, the evidence for the "isolation" of HL23V surpassed that of
HTLV-I and HIV in at least two aspects. Unlike HIV, Gallo's group: (a)
reported the detection of reverse transcriptase activity in fresh,
uncultured leucocytes;(34) (b) published an electron micrograph of
virus-like particles banding at a sucrose density of 1.16 gm/ml. (35)
Following the discovery of HL23V, some researchers attempted to
determine its prevalence utilising antibody tests (36) while others were
interested to determine the specificity of the antibody reactions. The
former included two of the best known HIV experts Reinhard Kurth and
Robin Weiss, and their colleagues who, for this purpose used "the simian
sarcoma-associated helper virus (SSAV) and the M7 strain of baboon
endogenous virus (BEV) to survey human sera for specific antibodies.
Also included is a virus (HL23V-1) originally isolated from cultured
peripheral blood leukocytes of a patient with acute myelogenous
leukemia. HL23V-1 was shown to comprise a mixture of two viruses, one
closely related to SSAV, the other to BEV" and found that "A survey of
human sera from healthy individuals revealed the presence of naturally
occurring antibodies that react in radioimmunoprecipitation assays with
proteins of mammalian type- C viruses" including the internal (gag) and
envelope (env) proteins of HL23V, SSAV and BEV and concluded, "The
serological studies presented here and by others provide indirect
evidence that the infectious mode of transmission remains a real
possibility in humans, and suggests that infection with an oncornavirus
[retrovirus] may be extremely widespread".(37) Three years later, in
1980, two research groups, (38,39) one from the Laboratory of Cellular
and Molecular Biology, National Cancer Institute and the other from the
Laboratory of Viral Oncology, Memorial Sloan-Kettering Cancer Center,
using the "viral glycoproteins", found that the antibodies present in
human sera which reacted with these proteins were "directed against
carbohydrate structures" and concluded that "The results are consistent
with the idea that the antibodies in question are elicited as a result
of exposure to many natural substances possessing widely cross-reacting
antigens and are not a result of widespread infection of man with
replication competent oncoviruses". In 1981 Gallo accepted the evidence
that the antibodies which reacted with proteins of HL23V were directed
not against the proteins "but against the carbohydrate moieties on the
molecule that are introduced by the host cell as a post-transcriptional
event, and which are therefore cell-specific and not
virus-specific".(40) This discovery was of such significance that today
nobody, not even Gallo, considers HL23V as being the first human
retrovirus, or even a retrovirus. In fact, in 1981 when Gallo and his
colleagues reported the presence in humans of antibodies to what he now
calls the first human retrovirus, HTLV-I, (according to Weiss, "The
first 'human' retrovirus to be isolated in 1971 was human foamy virus
(HFV) from a nasopharyngeal carcinoma line",(18)) the title of the paper
was, "Antibodies in human sera reactive against an internal structural
protein of human T-cell lymphoma virus".(40) In this paper Gallo and his
colleagues described the finding of antibodies to a "major internal
structural protein (p24) of HTLVCR" and claimed that such antibodies
were "specifically directed at HTLVCR proteins and not at cell-specific
determinants-in other words, the immunological reactions are not those
reported in human sera against animal virus glycoproteins which, lacking
virus specificity, are directed against the carbohydrate residues of the
glycoprotein".

(b) By 1989, researchers from New York showed that in Western blot
analyses, "the components visualized in the 120-160 kDa region do not
correspond to gp120 or its precursor but rather represent oligomers of
gp41". It was also shown that the WB pattern obtained is dependent on
many factors including temperature and the concentration of sodium
dodecyl sulphate used to disrupt the "pure virus". "Confusion over the
identification of these bands has resulted in incorrect conclusions in
experimental studies. Similarly, some clinical specimens may have been
identified erroneously as seropositive, on the assumption that these
bands reflected specific reactivity against two distinct viral
components and fulfilled a criterion for true or probable positivity.
The correct identification of these bands will affect the standards to
be established for Western Blot positivity: it may necessitate the
reinterpretation of published results".(41,42) (Little if any notice was
taken of this report!). Indeed, if, as it is claimed, HIV Western blots
are prepared from lysates of purified HIV virions, then it would be
impossible for p160 and p120 to be found in WB strips since: (i) All HIV
researchers agree with Montagnier and Gallo that gp160 is a precursor to
gp120 and gp41 and unlike the latter two proteins, is only found in
infected cells and not in mature particles; (ii) Although many EM have
been published of virus-like particles in non-banded material
nobody,(43,44) not even the CDC, (45) or Hans Gelderblom and his
colleagues who have most thoroughly studied these particles, has proven
the existence in the cultures of cell-free particles possessing knobs
(spikes). In one of his latest publications Gelderblom and his
colleagues have estimated that immediately after being released, "HIV
particles" possess an average of 0.5 knobs per particle but also pointed
out that "it was possible that structures resembling knobs might be
observed even when there was no gp120 present, i.e., false
positives".(46) It is accepted that gp120 is present only in the knobs
(spikes). Since there is no proof for the presence of knobs in the
cell-free particles, even immediately after release from the cell, it is
not possible for the gp120 to be present in the Western blot.

5.5 The "HIV pol protein", p31/34.

In 1987 Henderson isolated the p30-32 and p34-36 of "HIV purified by
double banding" in sucrose density gradients. By comparing the
amino-acid sequences of these proteins with Class II histocompatability
DR proteins, they concluded that "the DR alpha and beta chains appeared
to be identical to the p34-36 and p30-32 proteins respectively";(47)

5.6 The "HIV gag protein", p24

As far as Montagnier is concerned, p24 is THE HIV protein, and for at
least three years after the introduction of the "HIV" antibody test, a
p24 band found in the WB was considered by most laboratories, including
the CDC, as proof for HIV infection. At present there is ample evidence
that antibodies which react with p24 are ubiquitous in both human and
animal sera, which can only be interpreted that either p24, the
antibodies, or both, are non-HIV- specific or a significant proportion
of both humans and animals are infected with HIV. For example, if the
p24 band in the WB is considered proof of HIV infection then about 30%
of individuals who are transfused with HIV negative blood become
infected as a result.(48) Since, according to the AID vaccine Clinical
Trials Group, (49) "The presence of p24 band was common among low-risk,
uninfected volunteers and complicated the interpretation of the Western
blot test results", HIV infection should be common among healthy at no
risk individuals. In fact, because of such evidence, since 1987, with
perhaps only two exceptions, Montagnier and researchers conducting the
Multicenter AIDS Cohort Study in the United States, no laboratory
anywhere in the world considers a reaction between the p24 in the WB and
antibodies present in sera, as proof of HIV infection. Yet, when the
same reaction takes place between an antibody to the p24 of the WB and a
patient serum, it is considered proof of viraemia, and when between an
antibody to p24 and material present in a cell culture, the same
reaction is considered proof of HIV isolation!

Obviously, the detection of a protein, even if known to be virus
specific, in sera or even culture, does not constitute proof for
isolation or viraemia. That such a finding is non-specific can be best
illustrated by a few examples. In 1992, Jorg Shupbach, the principle
author of one of the first four 1984 papers published by Gallo's group
on HIV isolation, reported that the whole blood cultures of 49/60 (82%)
of "presumably uninfected but serologically indeterminate individuals
and 5/5 seronegative blood donors were found positive for p24".(50) If
p24 is an HIV protein then it must be present in all AIDS patients if
not all seropositive patients and not in persons not at risk of
developing AIDS. In 1989, David Ho and his colleagues used p24
measurements in serum and in cultures of non-infected cells cultured
with plasma from "infected" patients, to estimate active virus,
"infectious HIV-1", viraemia, viral load. The serum from 14/53 patients
whose plasma cultures were positive, was negative for p24. They
concluded, "Thus, plasma culture was more sensitive than serum p24
antigen measurement in detecting the presence of cell-free HIV-1 in
blood". They also reported that treatment with AZT for four weeks
induced "a 94 percent reduction in the load of cell-free virus".(51)
Even Jackson et al who claim an overall 98.3% "HIV isolation" rate, can
detect p24 in serum of 42% of AIDS patients, 37% of ARC patients and 17%
of asymptomatic seropositive individuals (52) which is a much lower rate
than in non-HIV infected organ transplant recipients. "In one kidney
recipient (the donor was negative for p24 antigen) who, 3 days following
transplantation developed fever, weakness, myalgias, cough and
diarrhoea, all "Bacteriological, parasitological and virological samples
remained negative [including HIV PCR]. The only positive result was
antigenaemia p24, positive with Abbot antigen kits in very high titers
of 1000pg/ml for polyclonal and 41pg/ml for monoclonal assays. This
antigenaemia was totally neutalizable with Abbot antiserum anti-p24...2
months after transplantation, all assays for p24-antigen became
negative, without appearance of antibodies against HIV. Five months
after transplantation our patient remains asymptomatic, renal function
is excellent, p24 antigenaemia still negative and HIV antibodies still
negative".(53) Using two kits, the Abbot and Diagnostic Pasteur, in one
study, p24 was detected transiently in 12/14 kidney recipients. Peak
titres ranged from 850 to 200 000 pg/ml 7-27 days post- transplantation.
Two heart and 5/7 bone marrow recipients were also positive, although
the titres were lower and ranged from 140-750 pg/ml. Disappearance of
p24 took longer in kidney (approximately 6 months) than in bone-marrow
(approximately 4-6 weeks) recipients. According to the authors: "This
may be related to differences in immunosuppression therapy". Discussing
their findings they wrote: "The observation of a 25-30kD protein binding
to polyclonal anti- HIV human sera after immunoblots with reactive sera
raises several questions. This protein could be related to a host immune
response to grafts or transplants...Its early detection after
transplantation might indicate the implications of immunosuppression
therapy...The 25-30kD protein could therefore be compared with the p28
antigen recently described with human T-cell- related virus
lymphotropic-endogenous sequence...The characterization of this 25-30kD
protein may represent an important contribution to the detection of
HIV-1-related endogenous retroviruses".(54) The disagreement between
Montagnier and Gallo about which proteins were actually "HIV" proteins
was not limited to gp41 but included p24. Montagnier always mentioned
that "no cross-reactivity existed between HIV p24 and other antibodies
including antibodies to HTLV- I, II". Until 1985 he also maintained that
there was "a very close homology between LAV and HTLV-III but an absence
of homology with HTLV-I and -II".(28) However, in 1985 he wrote, "We
have also compared the deduced amino-acid sequences of LAV proteins with
those of HTLV-I and other retroviruses and find no significant homology,
except for domains pol and gag which are generally conserved among
retroviruses".(55)

Gallo always maintained that homology exists between the HTLV-I, II
and HIV gag genes (56) and the many features shared by all "human
retroviruses" include "a small (p24/p25) major capsid protein; p24
cross-reactive antigenic determinant detected with either heterologous
(rabbit) antisera or human monoclonal antibodies".(57) Indeed, gag
stands for group specific antigens. As far back as 1974 Gelderblom and
his colleagues wrote, "While the virus envelope antigens are primarily
virus-strain specific, the bulk of internal proteins of the virion with
molecular weight (mw) between 10,000 d and 30,000 d are group-specific
(gs) for viruses originating in a given animal species (gs-spec.
antigens). The major protein constituent of mammalian C-type
oncornaviruses [retroviruses] with a molecular weight in the range of
30,000 d was found to possess, besides gs spec. antigen, an antigenic
determinant that is shared by C-type viruses of many mammalian species
including monkeys and was thus termed gs interspecies (gs-interspec.)
antigen".(58) In 1989 William Blattner, a well known HIV/AIDS expert
stated: "It may be feasible to use viral antigen probes to look for
cross-reactive antibodies, since certain viral proteins, particularly
the polymerase and gag proteins may be highly conserved between subtypes
of virus".(59) Thus, even if p24 were to be specific to retroviruses, it
cannot be HIV specific. If p24 detected in culture supernatants is a
component of similar particles, viral or non-viral, then in density
gradients all the p24 should be found at least in one band (fraction),
even if not at a density of 1.16 gm/ml. That this is not the case has
been demonstrated by Montagnier himself. In one experiment Montagnier
and his colleagues divided the density gradient into sixteen fractions.
The RT peak was found in fraction five and six, while the p24 and gp110
were present in all but three (1, 2, 3) fractions. (28)

5.7 The role of actin and myosin in particle budding.

There is no scientific reason to define a protein present in a cell,
culture supernatant, or even in material banding at 1.16 gm/ml in
sucrose density gradients as being retroviral on the basis that it
reacts with antibodies in AIDS patient sera, as Montagnier and Gallo's
groups did. According to Gelderblom, AIDS patient sera are
"polyspecific"(60,61) and at present there is ample evidence that these
sera react with a plethora of self and non-self antigens including
proteins of "non-infected" lymphocytes. Why then should they not also
react with the "HIV proteins", even if such proteins are cellular
proteins, or with a variety of recombinant or synthetic antigens? If the
proteins in the cultures/co-cultures of tissues derived from AIDS
patients and which react with AIDS patient sera are indeed retroviral,
then what are the proteins in the "non-infected" cells and supernatants
which Montagnier repeatedly reported to also react with AIDS patient
sera? On the basis of reactivity with AIDS patient sera, only 20% of the
proteins which band at 1.16 gm/ml can be considered "HIV proteins" and,
as the HIV/AIDS experts claim, without proof, are coded by "HIV
DNA".(47,62) Even if there was proof that pure (isolated) "HIV"
particles are present at 1.16 gm/ml, then all the proteins banding at
1.16 gm/ml should be embodied in such particles. However, since only 20%
of these proteins are "HIV" proteins, the question then arises, what is
the origin and role of the remaining 80% of the proteins in such
particles and by what genes are they coded? Why are only 20% of the
proteins viral and non-cellular? Why not all of them and vice versa? If
the gp41 protein present in the Western blot band and which reacts with
AIDS patient sera could be the ubiquitous protein actin, then why should
not one consider the p24 protein as being one of the light chains of
myosin, another equally ubiquitous protein especially given that: (a)
Matsiota, Montagnier and their colleagues at the Pasteur Institute have
shown that AIDS patients and those at risk have high levels of
antibodies to this protein;(63) (b) at present there is ample evidence
that the plethora of cellular proteins (á2 microglobulin, the à and á
chains of human lymphocyte antigen (HLA) DR, CD71, CD63, CD43, CD8, "the
major leukocyte adhesion receptors LFA-1 (CD11A/CD18) and CD44) which
are present in the "HIV particles", include actin and myosin.(64-68)
Indeed, in the last few years researchers from a number of institutions
expressed the view that actin polymerisation (or actin/myosin
interaction) "mediates HIV budding" and release. Researchers from New
York and Philadelphia found that colchicine treatment of
"MOLT4/HIV-1IIIB" cells, "induced lymphocyte polarization,
redistribution of F-actin into a pseudopod, and secretion of HIV from
the pseudopod", and that the particles were "observed exclusively on the
tip of the pseudopod". 65 Two of the studies which examined the role of
actin and myosin in "HIV particle" budding and release are by
researchers from Japan. In one publication the authors concluded, "Since
F-actin is essential for maintaining cell-shape and cellular function,
polarization of F- actin might change the cell membrane configuration or
cell fragility, which may be essential for HIV release".(67) In the
other study, the authors "demonstrated that myosin and actin are
colocalised at the budding site of viral particles. In particular,
myosin was concentrated on the same area of the plasma membrane as the
dense spots of the viral particles. In contrast, actin was widely
distributed on the plasma membrane and was always found in areas where
viral particles were present". They concluded, "actin might participate
with myosin in an active process leading to the release of viral
particles from the membrane". Because these researchers, like most
others, are of the opinion that "the initiation of a myosin-actin
interaction requires an increase in free intracellular calcium", they
have performed a preliminary experiment using two calcium chelators,
one, BAPTA which they consider chelates only intracellular free calcium
and the other, EGTA, which in their view chelates only the free calcium
on the outer side of the cell. They found that "HIV-1 release was
suppressed most pronouncably when both" the inner and the outer free
calcium was chelated, and that inhibition was stronger with the outer
chelater than the inner. "From these results, we suggest that [Ca2+]o
might enter the cell by the stimulation of viral budding itself at the
budding site...it may be difficult to detect an increase of
[Ca2+]i...because the budding mechanism is going on continuously and
slowly in a very narrow region without any synchronization".(64)

At present evidence also exists that: (a) there is an association
between the redistribution of polymerised actin, myosin and other
cellular proteins (glycoproteins) and many cellular processes including
budding unrelated to HIV release;(69-73) (b) polymerisation of actin,
actin-myosin interaction and cross- linking of polymers in general is
regulated by the redox state, oxidation leading to interaction;(74-76)
(c) both AIDS patients and cultures derived from AIDS patients are
subjected to oxidising agents. In fact, for the detection of "HIV",
proteins and particles the cell cultures must be stimulated (treated
with oxidising agents).(77)) Ten years ago Montagnier wrote, "Indeed,
LAV infection of resting T4 cells does not lead to viral replication or
to expression of viral antigen on the cell surface, while stimulation by
lectins or antigens of the same cells results in the production of viral
particles, antigenic expression and the cytopathic effect".(78) (d) in
the presence of antioxidants no "HIV" phenomena can be
observed.(77,79,80) In a study presented at this year's International
AIDS Conference, researchers from Rome reported, "The results obtained
using 3-ABA, NAC [antioxidants] and a combined treatment 3-ABA/NAC given
together seem to confirm the role of intracellular redox balance in the
modulation of the HIV expression. In fact, a significant reduction in
the number of viral particles was observed in cultures which have
received the combined treatment with NAC/ABA".(81)

Given the above data, may one be tempted to speculate that the "HIV"
particles and proteins are nothing more than "non-viral material
altogether", induced by the agents to which the AIDS patients and
cultures are exposed?

CONCLUSION

The statement "antibodies against Montagnier's HIV strain-the global
standard of all "HIV tests"", presumes proof of: (a) the existence of
more than one "HIV strain", including one of Montagnier's. Such evidence
can be obtained only by isolating the retrovirus. However, Montagnier's
evidence does not prove the isolation of a retrovirus; (b) the existence
of "HIV" specific immunogenic proteins. Again, such proof can be
obtained only by isolating the retrovirus; (c) antibodies specifically
induced by HIV infection. It is true that for detection of such
antibodies one does not need to use HIV or the HIV immunogenic proteins.
For example, serological tests for both infectious mononucleosis and
syphilis employ antigens derived from horse red blood cells and ox heart
respectively but nonetheless predict infection with Epstein-Barr virus
and Treponema pallidum. However, the only way to prove that "HIV
antibodies" are directed against "HIV", that is, the only way to use the
antibody test to prove HIV infection, is to present evidence which
proves that the antibodies are HIV specific. Such proof can be obtained
only by using HIV isolation as a gold standard. Since this has not been
done it is not possible to say that "the global standard of all "HIV
tests"" proves HIV infection.

6. "HIV DNA"

In debating the proof for the existence of a unique, exogenous
retroviral agent one cannot adopt as an initial premise ("Full- length
HIV-1 and HIV-2 DNAs...") that is contingent upon proof of the arugment
("ergo...HIV exists and has been isolated"). The a priori designation of
a particular fragment of DNA as "HIV DNA" merely begs the question under
consideration.

6.1 MINIMUM EVIDENCE REQUIRED TO PROVE THE EXISTENCE OF HIV
DNA

If "HIV DNA" is the genome of a unique retroviral particle then the
most basic requirement is proof for the existence of a unique molecular
entity "HIV DNA", that is, unique fragments of DNA identical in both
composition and length in all infected individuals. The claim that a
stretch of RNA (cDNA) is a unique molecular entity which constitutes the
genome of a unique retrovirus can be accepted if and only if it is shown
that the RNA belongs to a particle with the morphological, physical and
replicative characteristics of a retroviral particle. Proof of these
properties can only by obtained by isolating the putative viral
particles, that is, by obtaining them separate from everything else,
extracting the nucleic acids and demonstrating that such particles are
identical (their constituents including their nucleic acids are
identical) and infectious. The correct procedures, now having been used
for over half a century to achieve this proof, require demonstration
that: 1. In "infected" cell cultures (cocultures) there are particles
with a diameter of 100-120nM containing "condensed inner bodies (cores)"
and surfaces "studded with projections (spikes, knobs)"; (82) 2. In
sucrose density gradients the particles band at a density of 1.16 gm/ml;
3. At the density of 1.16 gm/ml these is nothing else but particles with
the morphological characteristics of retroviral particles; 4. The
particles contain only RNA and not DNA and that the RNA consistently has
the same length (number of bases) and composition no matter how many
times the experiment is repeated; 5. When the particles are introduced
into secondary cultures, but mindful of the critical caveat discussed
below: (a) the particles are taken up by the cells; (b) the entire RNA
is reverse transcribed into cDNA; (c) the entire cDNA is inserted into
the cellular DNA; (d) the DNA is transcribed into RNA which is
translated into proteins; 6. As a result of 5 the cells in the secondary
cultures release particles into the culture medium; 7. The particles
released in the secondary cultures have exactly the same characteristics
as the original particles, that is, they must have identical morphology,
band at 1.16 gm/ml and contain the same RNA and proteins.

The caveat is that while the introduction of the majority of
infectious particles into cell cultures and subsequent release of
similar particles is proof that such particles are indeed infectious,
this is not the sufficient case for retroviruses. The basis of this
exception is the fact that "one of the most striking features that
distinguishes retroviruses from all other animal viruses is the presence
in the chromosomes of normal uninfected cells, of genomes with those of
infectious viruses".(83) In fact, a cell may contain the genome of many
retroviruses. As far back as 1976 retrovirologists recognised that "the
failure to isolate endogenous viruses from certain species may reflect
the limitations of in vitro cocultivation techniques".(84) In other
words, the finding of a retrovirus in both the primary and secondary
"infected" cultures/cocultures is not proof that the cells have been
infected with an exogenous retrovirus.

One way which will suggest but will not prove that the cells acquired
virus from the outside (exogenously acquired retrovirus, infectious
retrovirus) and have not assembled a retrovirus from information already
existing in normal cells (endogenous retrovirus) is to conduct
experiments that use controls, that is, to run in parallel with test
cultures/cocultures control cultures/cocultures. The only difference
between the test and control cultures should be the introduction of
particles into the test cultures. In other words, apart from the
introduction of particles, in every other respect control cultures must
be dealt with identically. For example: (a) because detection of RT and
retroviral genetic sequences and release of retroviral particles depends
on the metabolic state of the cells, the physiological state of the
cells used in the control cultures should be as close as possible to
those of AIDS patients; (b) because the mere act of co-cultivation alone
may lead to release of endogenous retroviral particles, if test cells
are cocultured, so should the cells used in control experiments; (85)
(c) extracts, even from normal unstimulated cells, when added to the
cultures may increase endogenous retroviral expression. (86) Because of
this, when cells are cultured with "HIV" (supernatant or material which
bands at 1.16 gm/ml), the controls must be cultured with similar
material from cell cultures originating from sick individuals with
illnesses similar to AIDS, that is, matched individuals who are
immunosuppressed; (d) the appearance of endogenous retrovirus can be
accelerated and the yield increased a million fold by stimulating the
cultures with mitogens,(87) mutagens, chemical carcinogens and
radiation.(88,89) If test cultures are exposed to or employ such agents
so should the controls; (e) since AIDS patients and those at risk of
developing the syndrome are exposed to strong oxidising agents,(79,90)
the control cells should also originate from such patients; (g) to avoid
observer bias and in the best interests of science, blind examination of
test and control cultures/cocultures should be performed.

6.2 EVIDENCE FOR THE EXISTENCE OF "HIV DNA"

6.2.1 In 1984, in the first of two papers, Montagnier and his
colleagues described the following experiment: "Because LAV can induce
T-cell fusion and because EBV [Epstein Barr virus] is known to have
fusion activity in B cells, we performed co-infection experiments of
unfractionated lymphocytes (B and T) with both viruses. It was hoped
that stable hybrids of LAV-infected T cells and of EBV-transformed B
cells would be formed and that such hybrids would be able to
continuously produce LAV. Several regimens were tried. The one that gave
rise to continuous productive infection of LAV was the following. Whole
lymphocytes of F. R. were first stimulated for 24 hours with Protein A
and then infected with and EBV strain, M81, derived from a
nasopharyngeal carcinoma. Five days later, half of this culture was
infected with LAV as described (1) and then divided in two subcultures:
one was cultured in medium lacking T-cell growth factor (TCGF:
interleukin-2), the other in medium containing TCGF. As expected, the
TCGF-fed culture produced LAV as detected by a peak of RT activity
appearing between day 12 (day 6 after LAV infection) and day 21 in the
supernatant. In contrast, the cells cultured in the absence of TCGF did
not yield any detectable RT...On day 19, at the time of decline of LAV
production, a subculture of the TCGF-fed cells received fresh T cells
from the same donor: these T cells had been activated for 3 days with
phytohemagglutinin (PHA)...Six days later (day 25), a new peak of RT
appeared, but contrary to the first infection, it was not transient...At
the time of the second LAV infection, large round cells transformed by
EBV could be readily seen in this culture, as well as in the control
culture not infected with LAV, indicating that immortalization of the B
cells by EBV had already occurred. The immortalized B-cell line was
termed RF8".(29) [Reference 1 to which Montagnier refers is the 1983
paper in which Montagnier et al described the first "isolation" of HIV
(see 5)]. In the second study, 200 ml of supernatant from the "HIV
infected" FR8 cells were banded in sucrose gradients, "Virus containing
fractions were pooled" and centrifuged. (It is not stated how they
determined the existence of "virus", in which band(s) (fraction(s))
"virus" was found, how many bands if any were found to have particles,
or why there were more bands than one (1.16 gm/ml) containing the
"virus"). The pellet was incubated with several substances, dATP, dGTP,
dTPP, dCTP including 32dCTP and an oligo(dT) primer. From the cDNAs thus
obtained, three clones "pLAV13, 75 and 82, carrying inserts of 2.5, 0.6
and 0.8 kilobases (kb), respectively, were characterized further. All
three inserts have a common restriction pattern at one end, indicative
of a common priming site. "The 50-base pair (bp) common HindIII-PstI
fragment was sequenced and shown to contain an oligo(dA) stretch
preceding the cloning dC tail. The clones are thus copies of the 3' end
of a poly(A) RNA. The specificity of pLAV13 was determined in a series
of filter hydbridization experiments using nick-translated pLAV13 insert
as a probe". Firstly, "using an adapted spot-blot technique" they tested
the pellet obtained from the supernatant of "LAV infected" normal
lymphocytes and CEM cells as well as non-infected lymphocytes. The
"infected" pellets were positive and the non- infected negative.
"Second, the probe detected DNA in the Southern blots of LAV-infected T
lymphocytes and CEM cells. No hybridization was detected in DNA from
uninfected lymphocytes or from normal liver". No details are given
regarding the method used to produce "infection", but it would appear
that the normal cells and the CEM cells were cultured with supernatant
from the FR8 cells, that is, the same supernatant they used to obtain
the probe! They concluded: "Together, these data show that LAV pLAV13
DNA is exogenous to the human genome and detects both RNA and integrated
DNA forms, derived from LAV-infected cells. Thus, pLAV13 is LAV
specific".(91)

6.2.2 In May 1984, Gallo and his colleagues published four
papers. To "isolate" HIV they used a leukaemic cell line which they
called HT. It is impossible to known with what tissues from AIDS
patients this cell line was cultured. Reading the May 1984 papers one
gets the impression that the HT cell line was cultured with concentrated
(supernatant) fluids originating from individual, AIDS patient,
stimulated T-cell cultures. Subsequently, the Gallo investigation found
the HT cell line was cultured with concentrated fluids pooled initially
from individual cultures of three patients and ultimately from the
individual cultures of ten patients.(92) The Gallo investigation found
this procedure to be "of dubious scientific rigor". One scientist
described the procedure as "really crazy".(93) In 1985, Gallo and his
colleagues wrote, "The H9/HTLV-IIIB cell line was derived from the human
T-cell line HT, following co- culture with T lymphocytes obtained from
several AIDS patients, and contains many different HTLV-III forms".(94)
The detection of reverse transcription of A(n).dT15 in the supernatant,
was considered proof the HT cells were infected with a retrovirus, HIV,
which originated from the patients' tissues. A clone, H9 of the HT cell
line was obtained "using irradiated blood of a healthy donor as a
feeder".(21) The H9 cells were cultured with supernatant from the "HIV"
infected HT cells. The H9 supernatant was banded in sucrose density
gradients and the material which banded at 1.16 gm/ml which, without
proof, Gallo and his colleagues considered to be synonymous with
retroviral particles, was "lysed with sodium dodecyl sulfate (SDS),
digested with proteinase K, and directly chromatographed on an oligo(dT)
cellulose column. The resulting polyadenylate [poly(A)]-containing RNA
was used as template to synthesize 32P-labelled complementary DNA (cDNA)
in the presence of oligo(dT) primers. The size of the resultant cDNA
ranged from 0.1 to 10 kb. When these labelled cDNAs were hybridised to
poly(A)-containing RNA purified from infected [that is, cells cultured
with the same supernatants as those from which the probe was obtained]
and uninfected H9 cells as well as other uninfected human cell lines,
only the infected H9 cells contained homologous RNA sequences as
evidenced by discrete RNA bands after Northern hybridisation. Figure 1
shows that cDNA preparations from HTLV-IIIB and HTLV-IIIZ gave identical
patterns, detecting species of about 9.0, 4.2, and 2.0 kb...These bands
are similar in size to those corresponding to genomic size messenger RNA
(mRNA) and spliced mRNAs of env and pX sequences previously observed in
cells infected with HTLV-I, consistent with the anticipated relatedness
of these viruses. Furthermore, viral mRNA bands of HTLV-II-infected
cells were detected with an HTLV-III cDNA probe and again the sizes of
the mRNA were like those with HTLV-I"!(56)

In another study by Gallo and colleagues, extrachromosomal DNA of
"infected" H9 cells was extracted and "assayed for its content of
unintegrated viral DNA" using the 32P-labelled cDNA as a viral probe.
"Unintegrated linear viral DNA was first detected after 10 hr [of
"infection"] and was also present at the subsequent time points. Figure
1 shows a Southern blot of the 15-hr sampling. A band of ~10 kilobases
(kb) in the undigested DNA represents the linear form of unintegrated
HTLV-III".(95) In yet another study Gallo and his colleagues reported
that, "Since the HTLV-III provirus was found to lack Xba I restriction
sites, a genomic library was constructed by using Xba I-digested
H9/HTLV-III DNA, and this was screened with an HTLV-III cDNA probe to
obtain molecular clones of full length integrated provirus with flanking
cellular sequences. Fourteen such clones were obtained from an enriched
library of 106 recombinant phage, and two of these were plaque-purified
and characterized. Figure 1 illustrates the restriction maps of these
two clones, designated ^HXB-2 and ^HXB-3. The overall length of the
HTLV-III provirus is approximately 10 kilobases...To determine whether
the HTLV-III genome contains sequences homologous to normal human DNA,
the viral insert of ^XB-2...was isolated, nick translated and used to
probe HTLV-III-infected and uninfected cellular DNA. Under standard
condition of hybridization...this probe hybridized to DNA from
H9/HTLV-III cells as well as other HTLV-III-infected cells, but not to
DNA from uninfected H9 cells, uninfected HT cells (the parent line from
which H9 was cloned), or normal human tissues (data not shown). This
finding is in agreement with the results of other experiments in which
the unintegrated (replicative intermediate) form of HTLV-III was used as
a probe and demonstrates that HTLV-III, is an exogenous retrovirus
lacking nucleic acid sequences derived from human DNA".(96)

6.2.3 In 1984, Levy and his colleagues cultured PBMC from
patient suffering from Kaposis'sarcoma with IL-2, polybrene and PHA. The
supernatant was tested for RT, the cells for reaction with serum from
the Pasteur Institute patient BRU and "some cultures were examined for
virus by electron microscopy". The finding of a positive result with
"any of these tests" was considered proof of virus isolation. The
supernatant from one of these cultures was "inoculated into fresh human
PMC stimulated 3 days before with phytohemagglutinin". Within 6 days the
supernatant of this culture had high RT activity and this was said to
represent "the virus isolate ARV-2".(97) The HUT78 cell line was
cultured with "ARV-2". In the HUT78 "Virus production was monitored by
measuring reverse transcriptase activity". When there was maximum RT
activity, the supernatant was centrifuged and the resuspended pellet,
after treatment with DNAase, was centrifuged in sucrose gradients. The
nucleic acid from each fraction was electrophoresed on agarose gel. The
region in the gel containing an "~9kb RNA species was cut out" and used
to obtain "a radioactive cDNA probe". The DNA from the HUT78 cell line
cultured with "ARV-2" was digested with restriction enzymes,
electrophoresed in agarose gel and Southern blotted using the
"radioactive cDNA probe". "No specific bands were detected in several
digests of DNA from uninfected cells...whereas bands were seen in
infected cells...undigested DNA from infected cells contained a species
at 5.5 kb, a faint species at 6kb and a broad band at the exclusion
limit of the gel (>15kb). We suggest that the DNA species 5.5kb and 6
kb represent unintegrated viral DNA in a circular configuration
containing respectively one and two long terminal repeats (LTRs); the
upper broad band (>15kb) represents provirus integrated into the host
cell DNA". In an additional experiment "whole-cell DNA from cells
infected with ARV-2 was partially digested with ECORI; 9-15 kb cell DNA
was cloned into an EMBL-4 bacteriophage ^ vector and recombinant phage
were identified with the virus-specific cDNA probe". Among the
recombinant phage obtained were ^-9B and ^-7A, each of which was 9.5
kb.(98)

6.2.4 SUMMARY AND DISCUSSION

It is obvious that although Montagnier, Gallo and Levy and their
respective colleagues refer to virion or virus particles purification or
isolation, none of these groups have presented evidence for the
isolation of retrovirus particles or even the isolation of virus-like
particles, the first and absolutely necessary step in proving the
existence of a retroviral genome. (At the time of writing, neither has
any other group of HIV/AIDS researchers). Finding some RNA which bands
at 1.16 gm/ml, selecting from it a poly(A) rich fraction, or a fragment
of a given length, even if always found to be the same length and
sequence, and referring to it as HTLV-III, LAV, ARV does not constitute
such proof. It must be stressed that even if the RNA is incorporated in
a particle which in sucrose density gradients bands at 1.16 gm/ml, this
is still not proof that it is retroviral RNA. According to John Coffin,
one of the best known experts on the retroviral genome, there are
particles "with a full complement of viral proteins, but the particles
contain a collection of cellular RNAs and only about 1% genomic
RNA...assembly of particles does not require the genome...in its absence
other RNA molecules may be substituted".(83) It is important to note
that although all groups, Montagnier's, Gallo's and Levy's refer to the
material from the culture supernatants which in sucrose density
gradients bands at 1.16 gm/ml as viral particles, virions, and to the
RNA and proteins at that density as "particle-associated" RNA or
proteins, not one of the groups presented evidence for the existence at
this density of any particles, retroviral-like or otherwise, pure
(isolated) or otherwise. Instead these researchers cultured lymphocytes
from AIDS patients and stimulated (activated) them with a wide variety
of agents. Reverse transcription of A(n).dT15 in the culture supernatant
was considered proof for infection with a retrovirus or even proof of
isolation. Supernatants from these cultures were introduced into
cultures of leukaemic or transformed cell lines. With the supernatants
from these cultures they performed two types of experiments: (a) The
supernatants were banded in sucrose density gradients. At the 1.16 gm/ml
band (and sometimes at other band(s), at least in Montagnier's group
experiments, this is not made clear), they found fragments of RNA of
certain lengths (although no two had the same length) or were rich in
adenine, (poly(A) rich fragments), and called these "HIV RNA", the "HIV
genome". Using a (dT) primer the "HIV RNA" was transcribed into a
complementary DNA (cDNA); (b) The supernatants were introduced into
another set of the transformed and leukaemic cell lines as well as into
stimulated cultures of normal T-cells. The DNA from these cells, as well
as the DNA from the cultures to which no supernatant was added, were
hybridised using probes from the cDNA. Positive results were obtained
only with the DNA from the cells to which the supernatants were added.
This evidence was interpreted as proving that the "HIV DNA", the
retrovirus, originated from the AIDS patients and in fact that these
patient acquired it from the outside, that is, the retrovirus was
exogenous.

There are many problems associated with these experiments and their
interpretation. Among the many questions their conclusion raises the
most obvious are: 1. HIV is said to be a retrovirus and retroviruses are
particles which contain among other things, RNA. How then is it possible
to claim that the RNA which banded at 1.16 gm/ml, "HIV RNA", is the
genome of a retrovirus without proof that it is a constituent of a
particle, viral or non-viral which bands at this density? 2. RT is not
specific to retrovirus and in fact A(n).dT15 can be reverse transcribed
by all cellular DNA polymerases à, á and y. Is it possible then to
consider reverse transcription of A(n).dT15 as proof for HIV isolation
or even detection of a retrovirus? Even if the process of reverse
transcription is specific to retroviruses, can the detection of a
process ever be considered proof for the isolation of an object, in this
case, retroviral particles? 3. cell culture supernatants will contain
both DNA and RNA including some enclosed in cellular debris (fragments)
especially if cellular viability is not one hundred percent as is the
case in cultures used by the three groups. The RNAs may include
messenger RNA (which is adenine rich), as well as high molecular weight
heterogenous nucleic RNA. These RNAs, in addition to having high
molecular weight and heterogeneity in size, also have poly(A), with the
poly(A) attached at the 3' end of the molecule, and may be RNAase
resistant. Actinomycin, inhibits its synthesis and also interferes with
its proper processing and breakdown.(99) From animal virology it is also
known that non-retroviral RNA and DNA also bands at 1.16 gm/ml.(100) How
is it then possible to claim that just because an RNA bands at 1.16
gm/ml and is adenine rich or has a certain length, it is "HIV RNA"? If
this RNA is "HIV RNA", then what is the other RNA and the DNA which also
bands at this particular density? If the latter are cellular why not the
poly(A)RNA as well? 4. By definition, retroviruses are infectious
particles which contain only RNA. When they enter a cell the RNA is
reverse transcribed into DNA, which is then integrated into cellular DNA
as a provirus, which means that "HIV DNA" will be present only in the
cell and nowhere else. Yet many HIV experts including Gallo have shown
that both the supernatants of "infected" cell cultures and the "HIV
particles", that is, the material which bands at 1.16 gm/ml, contains
"HIV DNA" which "may integrate directly into the host chromosomal
DNA".(101-103) The question then arises, is the "HIV DNA" the result of
"HIV RNA" reverse transcription or is it vice versa? 5. It is accepted
that the HIV RNA is localised in a condensed core surrounded by a
"lipid-bilayered envelope derived from the cellular membrane of the host
cell, studded with virally encoded gp120 and myristylated protein, p17.
The so-called core-envelope link (CEL) attaches the core to the
envelope".(103) One of the best know facts in biology is that condensed
cores (chromatin) is transcriptionally inactive. This is one of the
reasons why viruses, including retroviruses, to multiply, must first
enter cells where their chromatin is decondensed. However, in a paper
published in 1993 by Hui Zhang and colleagues including Poiesz, from
Suny Health Science Center at Syracuse, New York, wrote: "We have shown
that in the absence of detergent, large amounts of DNAase-resistant
viral DNA can be synthesized within intact HIV-1 virions, indicating
that this phenomenon is not dependent on perturbation of the viral
envelope. [Not to mention decondensation of chromatin]. Nascent viral
DNA synthesis also occurred in purified virions incubated at 37ø in
cell-free human physiological fluids including seminal plasma, breast
milk, and fecal fluids"(103) This means that either (i) the "intact
HIV-1 virions" perform a function that no other biological system with
very condensed and protected chromatin can perform or (ii) the "HIV RNA"
found in the supernatants or in the "purified virions" is present in an
unembodied form or (iii) the "HIV RNAs" are de novo synthesised in the
cell cultures (see 6.3.5); 6. At present there is ample evidence that
any RNA or DNA present in the supernatant, irrespective of its origin,
especially when cells are stimulated by polycations and oxidising
agents, will be taken up by the cells (see 7.1). How is it then possible
to claim that a positive hybridisation signal in cells cultured with the
same "HIV DNA" containing supernatant as the supernatant from which the
"HIV DNA" probe originated but not in other cells is proof that the "HIV
DNA" is the genome of an exogenous retrovirus? 7. The first, absolutely
necessary step in proving that the "HIV DNA" originated from the
lymphocyte cells of AIDS patients and those at risk, is to perform
hybridisation experiments using the DNA of their fresh, uncultured
lymphocytes and the "HIV DNA" as a probe. It is hard to understand why
neither Montagnier's nor Levy's group reported such experiments. Gallo's
group did and the results were negative (see 6.4.4). How is it then
possible to claim that "HIV DNA" is the genome of an exogenous
retrovirus which originated from AIDS patients and those at risk? 8.
Reading the seminal paper on HIV isolation entitled "Detection,
Isolation and Continuous Production of Cytopathic Retroviruses
(HTLV-III) from patients with AIDS and Pre-AIDS", one gets the
impression that the leukaemic HT cell line which Gallo, Popovic, and
their colleagues used was a new cell line and one which they
established. The Gallo inquiry revealed that the HT (H9) cell line is
the same as that used by Levy's group, HUT78, a leukaemic cell line
established in another laboratory. However, the abundant evidence for
the existence of endogenous human retroviruses has largely been obtained
from experiments on leukaemic and transformed cells. Evidence exists
that both H9 and EBV-transformed B lymphocytes release retrovirus-like
particles even when not "infected with HIV".(104) Furthermore, the HUT78
(H9) cell line was established from a patient with "malignancies of
mature T4 cells", a disease which, according to Gallo, is caused by the
exogenous retrovirus, HTLV-I. Indeed, as far back as 1983, he claimed to
have shown that the HT (H9) cell line contained HTLV proviral
sequences.(105) According to some American researchers, EBV- transformed
normal human peripheral blood B lymphocytes contain HTLV-I related
transcripts.(106) Since all retroviral particles by definition band at
1.16 gm/ml, assuming that all the groups had a retrovirus at this
density, how is it possible to claim that the retrovirus originating
from the HUT78 and EBV-transformed B- lymphocytes is a new retrovirus
HIV, and not one which was already present? Can one claim that the "HIV
RNA" and thus the probes and primers originating from it are the RNA and
probes and primers of a unique exogenous retroviral genome? 9. The
biological dogma states that DNA is synthesised on a DNA template, RNA
on a DNA template, and proteins on an RNA template. In other words, the
only way for a cell to acquire new nucleic acid entities is for them to
be introduced from the outside, exogenously either from another cell
type, an infectious agent or a synthetic nucleic acid. If the biological
dogma is correct then the "HIV RNA", be it a cellular or viral molecular
entity, should have originated either from the patients' lymphocytes or
the transformed and leukaemic cell lines. However, when "HIV cDNA" was
used a probe, not one of the groups reported positive hybridization
results from any of the cells, not even from the lymphocytes of AIDS
patients. The question then arises, does a unique molecular entity, "HIV
DNA" exist? What does it mean and from where did it originate?

6.3. SPECULATIONS ON "HIV DNA"

If one wishes to speculate on the nature and origin of RNA (cDNA)
derived from the cultures containing tissues of AIDS patients and those
at risk, and which bands at 1.16 gm/ml, there are many possibilities
including:

6.3.1 Although to date no such evidence exists, it is possible
that the stretch of RNA, presently called "HIV RNA", is the genome of an
exogenous retrovirus, HIV. However, for this to be considered proven in
addition to satisfying all the requirements in 6.1 one must also show
that: (i) the unique stretch of RNA can be obtained only from cultures
of particular individuals; (ii) when the RNA (or cDNA) is used as a
probe to test fresh, uncultured lymphocytes, a positive test is obtained
only from the fresh cells of individuals who also have a positive
culture; (iii) that in animals or humans, the retrovirus is horizontally
(animal to animal, person to person) transmitted.

6.3.2 The genome of an endogenous retrovirus, that is, a
stretch of RNA with a corresponding DNA template present in the cellular
DNA of uninfected animals and which is passed from generation to
generation vertically (from parents to offspring via the germ cell line)
and which under certain conditions can be expressed and incorporated
into retroviral particles. For many decades it has been known that
animal DNA contains sequences "closely related or identical with those
of infectious viruses". However, the human genome was considered to be
an exception and as late as 1994, both Gallo and Fauci were of the
opinion that "...there are no known human endogenous retroviruses".107
In fact, in the 1970s and in the 1980s after Gallo's claim of the
discovery of HL23V, HTLV-I and later HTLV-II, and especially after
Montagnier's claim of the discovery of HIV, considerably greater
interest was engendered in retroviruses with the result that it became
"increasingly clear that the DNA of man, like that of other vertebrates,
contains many integrated retroviral genomes", (25,108) and that in many
cases the genes are expressed, "including mRNA transcripts related to
full-length endogenous retroviral DNA" (109,110) with open reading
frames for the gag, pol and env proteins.111 By 1987, many researchers
reported the expression of the genome of the human endogenous
retrovirus, HERV-K, homologous to the mouse mammary tumor virus (MMTV).
"In several cell lines, HERV-K genome was expressed as an 8.8 kilobase
poly(A)+ RNA which appears to be the full-length transcript of this
genome". When the human breast cancer cell line T47D was "grown in RPMI
1640 supplemented with 10% fetal calf serum, HERV-K genome expression
was slight". However, when the cells were treated with estradiol and
then progesterone, they produced "retroviruslike particles and soluble
protein sharing antigenic determinants with MMTV env gene product".(112)
In support of their thesis "that a human endogenous RT might mediate
gene movements leading to leukemia and cancer", researchers from
Hahnemann University, Philadelphia, including David Gillespie, a long
time collaborator of Gallo "demonstrated the presence of a reverse
transcriptase-like enzyme in retroviral particles from patients with
essential thrombocythemia, polycythemia vera, and chronic myelogenous
leukaemia. It was subsequently shown that the human genome contains 50
copies of HERV-K. HERV-K is a human endogenous class I retroviral
element that contains gag, pol and env open reading frames...as well as
intact LTR regions...Expression of a 9 kb genomic HERV-K RNA transcripts
were detected in human cell lines...We were able to show for the first
time the expression of HERV-K pol gene in human blood leukocytes. The
HERV-K pol gene was expressed in peripheral blood cells from two sets of
non-leukemic individuals. The first set consisted of 7 normal donors,
while the second set consisted of 3 patients with PV, all of which
expressed HERV-K pol gene. Five different nucleotide sequences were
obtained from the 7 normal donors. Four of the 5 normal sequences
contained heterogenous open reading frames for pol as detected by both
RT-PCR and RNAase protection. Unlike normal donors which randomly
express HERV-K proviruses, analysis of HERV-K pol from PV patient showed
selective expression of a restricted family of related proviruses".(113)
By 1995, Gallo admitted that the human cell does contain retroviral
genomes but he still insisted they are defective, "Retroviruses are
transmitted either genetically (endogenous forms) or as infectious
agents (exogenous forms). As do many other animal species, humans have
both forms...The DNA of many species, including humans, harbor multiple
copies of different retroviral proviruses. The human endogenous proviral
sequences are virtually all defective, and comprise about one percent of
the human genome".(114) The view regarding defectiveness is not shared
even by Reinhard Kurth who, with his colleagues, have extensively
studied the human endogenous retroviruses (115) and have shown that
HERV-K sequences are transcribed and that a human teratocarcinoma cell
line, GH, which contains these sequences, when examined by EM was found
to produce "human teratocarcinoma-derived retrovirus (HTDV) particles".
By 1993 Kurth and colleagues reported that in the GH cell line, "Four
viral mRNA species could be identified, including a full-length mRNA.
The other three subgenomic RNAs are generated by single or double
splicing events...Sequence analysis of expressed HERV-K genomes revealed
non-defective gag genes, a prerequisite for particle formation. Open
reading frames were also observed in pol and env. Antisera raised
against recombinant gag proteins of HERV-K stained HTDV particles in
immunoelectron microscopy, linking them to the HERV-K family".
Discussing their findings they wrote: "In Northern blots, expression of
HERV-K could only be demonstrated in teratocarcinoma cell lines but not
in other human lines. Preliminary RT PCR studies suggest, however, that
HERV-K may be expressed in many if not all human cells at levels to low
to be detectable in Northern blots. The basis of the significant
quantitative differences in expression between teratocarcinoma cells and
other cell lines is not clear. It is intriguing to speculate that a
cellular factor(s) may regulate the synthesis of HERV-K mRNA depending
on the cell type or the state of differentiation. In this context, it
should be remembered that other retroid elements [ERV-9, RTLVL-H,
LINE-1] are also preferentially expressed in human teratocarcinoma
cells".(116) It is of interest to note that Montagnier and his
colleagues reported their "HIV genome" from a transformed cell line,
that Levy and colleagues' HUT78 cell line is a human leukaemic cell line
and that Gallo and colleagues' H9 cell line is none other than HUT78,
and thus must have HTLV-I as well as endogenous retrovirus. It is
equally important to note that although Kurth et al found no sequence
homology between HERV-K and "human T-lymphotropic virus" or HIV, many
researchers reported HTLV-I sequences in the human genome including in
cell lines derived from teratocarcinoma.

In a paper published in 1985 researchers from a number of
institutions in the USA including the Laboratory of Tumor Immunology and
Biology, National Cancer Institute, Bethesda, it was reported that
"Human DNA contains multiple copies of a novel class of endogenous
retroviral genomes. Analysis of a human recombinant DNA clone (HLM-2)
containing one such proviral genome revealed that it is a mosaic of
retroviral-related sequences with the organization and length of known
endogenous retroviral genomes. The HLM-2 long terminal repeat hybridized
with the long terminal repeat of the squirrel monkey virus, a type D
virus. The HLM-2 gag and pol genes share extensive homology with those
of the M432 retrovirus (a type A-related retrovirus), mouse mammary
tumor virus (a type B retrovirus), and the avian Rous sarcoma virus (a
type C retrovirus). Nucleotide sequence analysis revealed regions in the
HLM-2 pol gene that were as much as 70% identical to the mouse mammary
tumor virus pol gene. A portion of the putative HLM-2 env gene
hybridised with the corresponding region of the M432 viral genome". The
pol region of HLM-2 showed homology with HTLV-I which, according to the
authors. "is not endogenous to human cells but is transmitted
horizontally as an infectious tumor-inducing virus of humans".(117)

In 1987 researchers from Canada reported the finding of a "Human
Endogenous Retroviruslike Genome with Type C pol sequences and gag
sequences related to the Human T-cell Lymphotropic Viruses", HTLV-I and
HTLV-II.(118) In 1989 researchers from the Department of Biochemistry,
New York University showed that "human DNA contains a wide spectrum of
retrovirus-related reverse transcriptase coding sequences, including
some that are clearly related to human T-cell leukaemia virus type I and
II, some that are related to the L-I family of long interspersed
nucleotide sequences, and others that are related to previously
described human endogenous proviral DNAs. In addition, human T-cell
leukaemia virus type I-related sequences appear to be transcribed in
both normal human T cells and in a cell line derived from a human
teratocarcinoma".(119) In a paper published in 1989, researchers from
the USA summarised their experimental findings as follows: "Human T-cell
leukemia virus (HTLV) type I- related endogenous sequences (HRES) have
been cloned from a human genomic library. HRES-1/1 is present in DNA of
all normal donors examined. By nucleotide sequence analysis, HRES-1/1
contains two potential open reading frames capable of encoding a p25 and
a p15. A 684 flanking region 5' from the first ATG codon of p25 contains
a TATA-box, a poly-adenylation signal, a putative tRNA primer binding
site, and inverted repeats at locations which are typical of a
retroviral long terminal repeat...The HRES-1/1 genomic locus is
transcriptionally active in lymphoid cells", including EBV- transformed
normal human peripheral blood lymphocytes, leukemic cell lines, melanoma
cells and embryonic tissues.(106) In a paper published in 1992 by
researchers from Hungary and Britain entitled "Human T-cell lymphotropic
virus (HTLV)-related endogenous sequences, HRES-1, encodes a 28-kDa
protein: A possible autoantigen for HTLV-I gag-reactive autoantibodies",
the "presence of a human T-cell lymphotropic virus (HTLV)-related
endogenous sequence, HRES- 1, in the human genome was documented. The
HRES-1 genomic locus is transcriptionally active and contains open
reading frames...Antibodies to HRES-1-specific synthetic peptides were
noted in patients with MS, progressive systemic sclerosis (PSS), SLW,
Sjogren syndrome (SJS), and essential cryoglobulinemia (ECG). The data
suggest that HRES-1 may serve as an autoantigen and correspond to a
natural target of HTLV-I core protein-reactive autoantibodies".(120)

In the virological literature there is ample evidence which shows
that when a cell contains two proviruses, progeny may be found that
possess the genome of one but the structural proteins of either or both
viruses present. Conversely, the RNA may be viral but at least some of
the proteins may be cellular. In other instances, the particles do not
have a genome at all, or one or more genes are missing (genetically
defective viruses). The genetic mixing can be between viral genomes or
between viral and cellular genes.(83,121) According to distinguished
retrovirologists such as Weiss and Temin, new retroviral genomes may
arise by rearrangement of cellular DNA caused by many factors including
pathogenic processes, a view that proposes retroviruses as an effect and
not the cause of disease.(122,123) According to Varmus, "Retroviral
genomes recombine at high frequency (estimates range as high as 10 to
30% for each cycle of multiplication), and heterodimeric RNAs are
thought to be intermediates, with recombination taking place during
reverse transcription. Recombination appears to be strongly favoured by
homology, but joining also occurs occasionally between unrelated
sequences, e.g., during the latter phase of genetic transduction by
retroviruses. When viruses are grown in cells that contain related
endogenous proviruses, packageable transcripts from those proviruses may
participate in recombination reactions with the exogenous virus. This is
most dramatically revealed by the repair of deletion mutations in the
genome of an exogenous virus in a fashion that superficially resembles
gene conversion". In some animals proviruses have been acquired "during
recent breeding of the strains in the laboratory" and "in a few
instances, endogenous proviruses have been established or
increased in number during experimental observations"(121) (italics
ours).

As far back as 1974, based on the then available evidence, Howard
Temin proposed that the retroviral (ribodeoxyviruses) genomes originate
from "normal cellular components. The relationships between the
different ribodeoxyvirus groups reflect the relationships among the
cellular components from which the viruses evolved and the convergent
evolution of the viruses. In other words, there are relationships among
ribodeoxyviruses because the ribodeoxyviruses evolved from cells which
themselves had relationships deriving from common ancestors. A possible
mechanism of this evolution is described in Fig. 5". In the legend to
Fig. 5 Temin wrote. "A section of a cell genome becomes modified in
successive DNA (W) to RNA (-) to DNA transfers until it becomes a
ribodeoxyvirus genome. First, these sequences evolve as part of a
cellular genome. After they have escaped as a virus, they evolve
independently as a virus genome. The time scale may be millions of years
in germ-line cells and days in somatic cells".(122) Temin reinforced his
view in a more recent publication.(124)

In 1975, Gallo, Gillepsie and their colleagues wrote: "Even though
RNA of class II [exogenous] retroviruses shows minimal homology to
uninfected host cell DNA, hybridization of nucleic acids among class II
leukemia viruses from different species gives a pattern which is the
same as the phylogenetic relatedness among their natural hosts...We have
proposed that these and other results favor the interpretation that all
RNA tumor viruses are derived from cell genes, a proposal in agreement
with the virogene theory...By analysis of the RNA of viruses infecting
and replicating in a new host, evidence has also been obtained which
indicates that the genome of type C viruses can be substantially changed
by the host, probably by recombination with host DNA".(125) A few years
later, Coffin wrote: "The close relationship of virion proteins as well
as overall nucleic acid homology must mean that both exogenous and
endogenous avian tumor viruses [retroviruses] derive from a common
ancestor".(126)

In 1991 researchers from the New York University published a paper
entitled, "Evolutionary Implications of Primate Endogenous Retrovirus".
Discussing the presently available data they wrote, "A recent detailed
phylogenetic analysis of exogenous and endogenous retroviruses
(including retrotransposons) strongly suggests that a pool of endogenous
retroviral sequences periodically contributes to the generation of
exogenous viruses, and that the presence of endogenous primate
retroviruses is probably more directly related to exogenous viruses that
might have been thought".(127)

6.3.4 The "novel" RNA found in the cell culture supernatant
and the material from it banding at 1.16 gm/ml, the "HIV RNA", may have
nothing to do with a retroviral genome. It may be an RNA obtained by
transposition, that is, by certain replicating DNA sequences
(transposons) becoming inserted elsewhere in the genome, or by
retroposition, that is, by particular RNA (retrotransposons) first being
transcribed into DNA and then similarly being inserted into the genome.
Retroposition can "use cellular mechanisms for passive retroposition, as
well as retroelements containing reverse transcriptase". The
retroelements may be retrovirus-like elements or nonviral
elements.(128,129) Not only can retroposition "shape and reshape the
eukaryocytic genome in many different ways"(128) but the nonviral
retroelements may be similar to the retroviral elements. According to
Doolittle et al from the University of California, San Diego,"...the
entire group of reverse transcriptase-bearing agents, including
retrotransposons and genuine retroviruses, has recently been dubbed,
"retroids". Sequence comparisons by many other workers leave little
doubt that the reverse transcriptases of all the "retroids" considered
here are homologous, which is to say, the sequence resemblances are not
the result of chance or convergences. Our own comparisons confirm that
general notion, not only for reverse transcriptases, but also for the
ribonucleases, endonucleases and proteases, although it should be
understood that not all "retroids" contain all four enzymes...All of
these elements have additional features in common with retroviruses
including characteristic LTRs (long terminal repeats) and primer sites
that are complementary to various tRNAs. Like retroviruses, most contain
distinctive nucleic acid-binding and core particle proteins; in electron
micrographs there is a remarkable likeness to retroviral capsids...About
the only feature that regularly distinguishes many of these
retrotransposons from genuine retroviruses is the absence of an envelope
protein".(17)

6.3.5 Although half a century has passed since the Nobel
laureate Barbara McClintock discovered the phenomenon of transposition
which can lead to the appearance of new genotypes and phenotypes, at
present it is still generally accepted that any time one finds a
particular stretch of RNA in a cell, for example, in a T- lymphocyte,
unless RNA or DNA has been introduced from outside, all T-cells,
regardless of their physiological state or stresses to which they are
subject, will contain a corresponding stretch of DNA. In other words,
the DNA (genes) in a cell are invariant and all RNA molecules in the
cell are subservient to a matching length of DNA. However, according to
McClintock, the genome can be restructured and not only by
transposition. In her Nobel lecture of 8th December 1983, she said,
"rapid reorganisation of genomes may underline some species formation.
Our present knowledge would suggest that these reorganizations originate
from some "shock" that forced the genome to restructure itself in order
to overcome a threat to its survival...Major genomic restructuring most
certainly accompanied formation of new species". The "genomic shock"
which leads to the origin of new species may be "either produced by
accidents occurring within the cell itself, or imposed from without
such as virus infections, species crosses, poisons of various sorts, or
even altered surroundings such as those imposed by tissue culture.
We are aware of some of the mishaps affecting DNA and also of their
repair mechanisms, but many others could be difficult to recognize.
Homeostatic adjustments to various accidents would be required if these
accidents occur frequently. Many such mishaps and their adjustments
would not be detected unless some event or observation directed
attention to them...Unquestionably, we will emerge from this
revolutionary period with modified views of components of cells and how
they operate, but only however, to await the emergence of the next
revolutionary phase that again will bring startling changes in
concepts"(130) [italics ours and see this reference for examples].

In the 1980s a number of phenomena have been discovered which brought
startling changes in concepts including the following: Up until the late
1970s, the prevailing concept was that a discrete, contiguous stretch of
DNA is a structural gene encoding the genetic information to specify the
manufacture of a single protein, and that the linear sequence of the
nucleotides in this stretch of DNA corresponds directly to the linear
sequences of the RNA nucleotides and to the amino acids in the protein.
The first discovery which contradicted this belief was the discovery
that the DNA base sequences which coded for a given protein were not in
a continuous stretch of DNA but may be interspersed with other,
non-coding base sequences, that is, the genes are split,
"genes-in-pieces". A number of mechanisms have been postulated to
account for this observation. In one such explanation it is hypothesised
that the entire stretch of DNA is transcribed into a piece of RNA, then
the non-coding regions (introns) are excised and the coding regions
(exons) are spliced together to make the appropriate messenger RNA.(131)
There are no rules setting an upper limit on the number of introns in a
"gene", some genes may have up to sixteen or more introns. Nor are there
any rules regarding the length of introns, although in general, introns
are much longer than exons, the length of exons "peaking at about 40 or
50 amino acids...the shortest intron being 50 bases long, the longest
extending out to some 50.000 bp".(132)

According to Gilbert introns represent "hot spots" for recombination
and new genes can be created "through the coupling of exons by
intron-mediated recombination", "introns are lost and more complicated
exons are formed".(133) At present evidence exists showing that at least
some introns are mobile genetic elements, transposable elements, they
self-splice, they often contain reading frames capable of encoding a
protein including "regions of homology to reverse transcriptase
scattered over a roughly 250- amino acid stretch in the middle of each
intron ORF".(134) The discovery of split genes "shows that the genetic
apparatus of the cell is more complex, more dynamic than any of us had
suspected".132 Another strongly held view was the belief that all
cellular reactions and thus gene splicing were catalysed by a protein
enzyme. In the early 1980s it was found that RNA can cut, splice and
assemble itself, as well as assemble RNAs other than
itself.(135-138)

6.3.6 One of the strongest held views in biology is the belief
that nucleic acids have an inherent ability of instructing their own
synthesis and that nucleic acids cannot be synthesised in the absence of
a nucleic acid template. Manfred Eigen and his colleagues in Germany
conducted extensive theoretical and experimental work on molecular
self-replication.(139) In their experimental work they used the
bacterial virus (phage) Qá. In addition to its genome, a simple strand
RNA molecule of 4500 nucleotides, the virus has an RNA molecule of 220
nucleotides known as "Spiegelman's minivariant" which, like the genomic
RNA, is reproduced in cell-free laboratory systems by an enzyme called
Qá replicase. By mixing Mg2+ ions, the nucleoside triphosphates ATP,
GTP, UTP, CTP, Qá replicase and template RNA, they could obtain RNA
replication but a totally unsuspected finding was that even the absence
of the template, RNA was still synthesised. They performed many
experiments to prove this phenomenon and to exclude the possibility of
the presence of an initial RNA template and concluded, "Finally we were
convinced we had before us RNA molecules that had been synthesised de
novo by the Qá replicase enzyme. What was most puzzling, the de novo
product had a uniform composition which in each trial turned out to be
similar to or even identical with Spiegelman's minivariant". When the
template free mixture was then divided into several isolated
compartments where optimal conditions for de novo synthesis were
maintained they found that "each component had a uniform population of
de novo product, the products differed from compartment to compartment.
Further analysis revealed however that the different sequences were not
completely unrelated...There was a definite, uniform final product for
any set of experimental conditions, but here were as many different
optimal products as there were different experimental conditions. One of
the optimal products appeared to be Spiegelman's minivariant...Other
products of optimization were adapted to conditions that would destroy
RNAs, such as high concentrations of ribonuclease, an enzyme that
cleaves RNA into pieces...Some variants were so well adapted to odd
environments that they had a replication efficiency as much as 1000
times that of variants adapted to a normal environment...Any RNA formed
by noninstructed chemistry would be reproduced by template-instructed
chemistry at a rate proportional to the current RNA concentration. The
result would be exponential growth. Furthermore, even if only a single
template were formed initially by noninstructed synthesis, there would
soon be a host of different sequences because errors (point mutations,
insertions and deletions) would inevitably be made in the course of
replication. Hence in each generation there would be not only a larger
number of RNA strands but also a greater variety of RNA sequences. What
would happen then? Some of the mutants would be copied more rapidly than
others or would be less susceptible to errors in copying, and their
concentration would increase more rapidly. Sooner or later these
faster-growing mutants would take over...Hence the results of the
self-replication competition had to be the master sequence together with
a huge swarm of mutants derived from it and from which it had no way of
escape...We call this entire mutant distribution a quasispecies. It is
the quasispecies mutant distribution that survives the competition among
self-replicating RNAs and not just one master sequence or several
equivalent ones that are the fittest genes in the distribution. The
essence of selection them is the stability of the quasispecies".(140)
According to Eigen and his colleagues, the maximum length of an RNA
master sequence is of the order of 10,000 nucleotides.(139,141)

6.3.7 A basic principle of molecular biology is that the
primary sequence of RNA faithfully reflects the primary sequence of the
DNA from which it is transcribed. However, in the 1980s RNA editing,
"broadly defined as a process that changes the nucleotide sequences of
an RNA molecule from that of the DNA template encoding it", was
discovered. In the process a non-functional transcript can be
retailored, producing a translatable mRNA, or modify an already
functioning mRNA so that it generates a protein of altered amino acid
sequences. Sometimes editing is so extensive that the majority of
sequences in a mRNA are not genomically encoded but are generated
post-transcriptionally producing the "paradoxical situation of a
transcript that lacks sufficient complementarity to hybridize to its own
gene!".(142-144) According to Nancy Maizels and Alan Weiner from the
Department of Molecular Biophysics and Biochemistry at Yale University,
"the central dogma has survived hard times. The discovery of reverse
transcriptase amended but did not violate the central dogma of how genes
make proteins; introns qualified the conclusion that genes are
necessarily collinear with the proteins they encode; somatic
rearrangement of lymphocyte DNA called stability of eukaryotic genomes
into doubt...and catalytic RNA challenged the pre-eminence of proteins
and breathed new life into the ancient RNA world". However, the
discovery of RNA editing "could come close to dealing it a mortal
blow".(145)

6.3.8 CONCLUSION

The finding of a novel stretch of RNA or DNA and proteins in: (a)
lymphocytes of sick individuals or individuals who have been "shocked"
with agents such as physical or chemical mitogens, carcinogens or
oxidising agents in general as is the case with AIDS patients and those
at risk;(77,79,90) (b) lymphocytes in cultures or co-cultures (which
could lead to the appearance of hybrids) which have been additionally
"shocked" with sometimes multiple, similar agents; is not proof that the
given stretch of RNA comes from the outside, irrespective of its length,
the presence of poly(A) and number of ORF ("genes").

From Montagnier's, Gallo's and Levy's and their colleagues' evidence
it is not possible to conclude that the "HIV RNAs" they found are a "new
species" of RNAs induced by "shocking" the cells or by one or more of
the other phenomena which have come to light in the 1980s. Nor is it
possible to conclude that their RNAs are the genome of an exogenous
retrovirus as they did. However, a number of predictions can be made:
(a) If the "HIV DNA" is indeed the genome of an exogenous retrovirus
then: (i) there must be evidence to prove the existence of a unique
molecular entity "HIV RNA", and a corresponding fragment of DNA ("HIV
DNA") which has a unique length and unique nucleic acid sequences; (ii)
when the full length fragment of "HIV DNA" or "HIV cDNA" is used for
hybridisation studies all infected people should give a positive result.
(b) If the selected RNA which was found to band at 1.16 gm/ml, the "HIV
RNA", is the genome of a retrovirus which exists "in all of us",
endogenous retrovirus, then again evidence must prove the existence of a
unique molecular entity, "HIV RNA", ("HIV DNA"). When hybridisation
studies are conducted using the full length of the unique molecular
entity as a probe, positive results should be found "in all of us"; (c)
If the RNA found by the three groups, "HIV RNA", is the genome of a
retrovirus assembled de novo from DNA already existing in the cells, as
the result of in vivo or in vitro conditions, evidence must also prove
the existence of a unique molecular entity. When the whole length of the
unique fragment of nucleic acids is used as a hybridisation probe, a
positive result should only be found in cells which are subjected to
exactly the same in vivo or in vitro conditions as those from which the
"HIV RNA" at 1.16 gm/ml was obtained. When only fragments of "HIV RNA"
are used for hybridisation, the probability of finding a positive result
will increase; (d) If the "HIV RNA" is a unique non-viral molecular
species of RNA resulting from the transcription of a unique molecular
species of DNA then when the whole fragment of "HIV RNA", ("HIV cDNA")
is used a probe for hybridisation studies, a positive result should be
found only in the cells of the same type as those from which the "HIV
RNA" originated, in all individuals; (e) If the "HIV RNA" is neither the
genome of a retrovirus nor a faithful transcript of a fragment of DNA
present in the cells from which it has been obtained, but is the result
of the "shock" to which the cells have been exposed, either in vivo or
in vitro or both, or as a result of the phenomena discovered in the
1980s then: (i) since it is not possible to exactly reproduce the
conditions in vivo or in vitro to which the cells are subjected, it
would prove difficult if not impossible to always obtain a unique
molecular entity "HIV RNA", that is, to always obtain a fragment of RNA
or DNA of identical length and sequences; (ii) when the full-length
fragments of "HIV RNA" or "HIV cDNA" are used as hybridisation probes
there will be only a low probability of finding a positive result.
However, the probability will increase if only small fragments of the
"HIV RNA" or "HIV cDNA" are employed.

6.4. EVIDENCE THAT THE "HIV RNA" BELONGS TO AN EXOGENOUS
RETROVIRUS

The Montagnier, Gallo and Levy groups claimed that the special RNA
which they selected from the total RNA which in sucrose density
gradients banded at the density of 1.16 gm/ml was novel to the
lymphocytes and that in fact belonged to an exogenous retrovirus.
Although they did not present evidence to prove this assertion, the
possibility cannot be excluded that indeed this may have been the case.
Since at present their claim is generally accepted one would have
thought that by now they or other researchers should have been able to
provide ample confirmatory proof. This does not seem to be the case:

6.4.1 If the RNA originates from a retrovirus either
endogenous or exogenous then evidence must exist which proves that such
RNA is a constituent of particles which possess at least the most basic
morphological and physical features of retroviruses, that is, "a
diameter of 100-120 nm budding at cellular membranes. Cell released
virions contain condensed inner bodies (cores) and are studded with
projections (spikes, knobs)".(82) To date not only has nobody shown that
the "HIV RNA" belongs to such particles, there is no evidence that
particles of any kind are present in the material from cell
cultures/cocultures which bands at the retroviral density of 1.16 gm/ml
and from which the "HIV RNA" is selected. Furthermore, although
particles have been demonstrated in cultures, cultures contain many
different types of particles but none display BOTH principal
morphological characteristics, that is, "a diameter of 100-120 nm" AND
surfaces which "are studded with projections (spikes, knobs)".146

6.4.2 If the "HIV RNA" is the genome of an exogenous
retrovirus then, like the "exogenous animal retroviruses", one should be
able to find it in infected material without the necessity to revert to
the use of co-cultivation or mitogenically stimulated cultures. However,
none of the phenomena which are thought to prove the existence of HIV
can be detected unless one employs mitogens or co-cultures or both (and
sometimes additional "shock"), a fact accepted by both Montagnier and
Gallo.(78,147)

6.4.3 One cannot claim that "HIV RNA" is the genome of a
unique retrovirus, HIV, unless evidence is presented to prove that 'HIV"
is a unique molecular entity. By 1985 it was known that "the env genes
of ARV and HTLV-III differ by more than 20 percent" and that "the Gallo
group has sequenced another HTLV-III isolate and finds that it differs
from the first by about as much as ARC".(114,148) By 1986, Gallo and his
colleagues accepted that the "HIV genome" has a "far greater
variability" as "compared to HTLV" and in fact "The rate of genetic
change for the AIDS virus is more than a millionfold greater than for
most DNA genomes and may even be tenfold greater than for some other RNA
viruses including certain retroviruses and influenza A virus".(149) At
present it is accepted that "no two isolates are identical. Each isolate
contains many variants".(150) In one and the same patient the genomic
data in monocytes differs from that in T-lymphocytes.(151) There are
"striking differences" between the proviral DNA and cDNA in one and the
same PBMC sample "which could not be explained by either an artefact of
reverse transcriptase efficiency or template selection bias".(152) The
genetic data obtained in vitro do not correlate with the data obtained
in vivo, "to culture is to disturb".(153) According to the researchers
from the Pasteur Institute "an asymptomatic patient can harbour at least
106 genetically distinct variants of HIV, and for an AIDS patient the
figure is more than 108.(154,155) The "HIV genome" varies with time; in
one case where clones were obtained 16 months apart all the clones
detected in the second sample were distinct from the clones in the first
sample.(156) It is also accepted that up to 99.9% of the "HIV genomes"
may be defective.(157)

According to Levy, "The mechanism responsible for generating these
varying strains of virions is puzzling. One theoretical possibility is
that the unintegrated proviral copies of HIV that accumulate during
acute replicative infection can undergo efficient genomic recombination
leading to the evolution of infectious variants.(158) In Robin Weiss'
view, "the source of variation is the infidelity of reverse
transcription, which has no editing mechanism for transcriptional
errors", as well as "genetic recombination" especially when cell fusion
takes place.(159) By the late 1980s, researchers from the Pasteur
Institute concluded, "it is increasingly clear that it will be very
difficult to describe correctly the characteristics of HIV viruses using
single molecular clones". "It is evident that HIV, either in vivo or in
vitro, is extraordinarily complex and that a population-based approach",
a quasispecies approach as defined by Eigen, must be used to describe
HIV. They also added, "Even with a population-based approach, only small
regions of the HIV genome can be studied...Given such complexity and the
evident differences between quasispecies in vivo and in vitro, the task
of defining HIV infection in molecular terms will be
difficult".(153,160) The data which have been published since confirm
their conclusion. Prior to the 1990s, the HIV sequences were classified
as African and USA/European with sequence differences of 20-30 percent
between these two groups.(161) In the 1990s, HIV researchers started to
divide the "HIV genome" into subtypes A, B, C, D, E, etc. The basis for
this classification system is: "(a) subtypes are approximately
equidistant from one another in env (a 'star' phylogeny"); (b) the env
phylogenetic tree is for the most part congruent with gag phylogenetic
trees; (c) two or more samples are required to define a sequence
subtype". However, "Subtype naming problems have arisen for several
reasons. A small but not insignificant number of viral sequences are
hybrid, clustering with one sequence subtype in gag and another sequence
subtype in env, for example; or, to take another example, clustering
over different stretches with two or more subtypes in env...Naming
becomes problematic when highly divergent forms of a given subtype
arises: such forms are sometimes designated A', B', F', etc". It is
increasingly necessary to have sequence data from both gag and env
coding sequences when a new form or subtype is being claimed".(162)

By the middle of this year "at least ten" (A-J) prevalent major (M)
and a low prevalence, O, HIV-1 genotypes were described and new
genotypes are still reported.(8,163) According to researchers from the
Henry M Jackson Foundation Research Laboratory and Division of
Retrovirology, Walter Reed Army Institute, USA, "The great majority of
genotypic consignments for HIV-1 are based on subgenomic sequence
segments, typically encompassing 2% to 30% of the genome", and not by
comparisons of the whole genome. This is because, "it remains
impractical to obtain full length genomic sequences of HIV- 1 isolates
as a routine genotyping method, due to the low abundance of HIV-1
proviral DNA in clinical samples and virus cultures on PBMC substrate,
and to the relative inefficiency of the polymerase chain reaction when
amplicons become large". "The designation Human Immunodeficiency Virus
Type-1 (HIV-1) encompassed an unanticipated complexity of viral
forms".(163) According to researchers from the Los Alamos National
Laboratory, "while a subtype designation based on a gene or gene
fragment may be correct, recombination may have occurred. Therefore,
care should be taken to not over interpret the subtype designation. If
one is to discuss the subtype designation of viral isolates based on the
data presented here, they should be refer to the designation as 'B-like
over V3 loop region' rather than as 'subtype-B'".(164) One and the same
person may be "infected" with more than one subtype.(165) This means
that at present it is not possible to say what are the sequence
differences, both qualitative and quantitative, between different HIV-1
subtypes. Nonetheless, some suggestive data does exist. In 1993
researchers from several institutions "reported that in the A-G HIV-1
genotypes the intra-genotypic gag distances averaged 7% whereas the
inter-genotypic distances averaged 14%...The maximum level of
variability in gag is still well below that observed for the envelope
region of HIV-1".(166) Two HIV-1 strains, designated ANT70 and MVP5180
were isolated in 1987 and 1991 respectively from patients in Cameroon".
They were classified as HIV-1 subtype O. By 1994 evidence was presented
which "indicated that subtype O was endemic in Cameroon and Gabon".(167)
"DNA sequence analysis of MVP-5180 showed that its genetic organisation
was that of HIV-1, with 65% similarity to HIV-1 and 56% similarity to
HIV-2 consensus sequences. The env gene of MVP- 5180 had similarities to
HIV-1 and HIV-2 of 53 and of 49% respectively...Comparison of the
MVP-5180 amino acid sequence with that of the Gabon chimpanzee virus
showed similarities of 70, 78 and 53% in the gag, pol, and env genes,
respectively; similarities of 70, 76 and 51% to the Uganda HIV-1 (U455)
and of 54, 57 and 34% to the HIV-2 isolate D205 were found". The
researchers from Germany and Cameroon who conducted this study expressed
the view that "Even more divergent HIVs may exist. Such divergent HIVs
are likely to be transmitted by the usual routes (sexual and blood
contact and mother-to-infant transmission), leading to wider
distribution. They will have to be taken into account in vaccine
development and diagnostic test sensitivity and specificity".(168)
Indeed, this seems to be the case. Last year, David Ho and his
associates (169) studied an Australian patient with "primary infection".
"Since seroconverters generally harbor a relatively homogenous
population of viruses", they were surprised when they found that he was
"co-infected", "by multiple subtype B HIV-1...The average genetic
distances between group I and II, I and III, and II and III were 9.6,
16.5 and 18.4% respectively...One population of sequences was clearly
distinguishable from the others on the basic of phylogenetic analysis,
In addition, sequences suggesting recombination between two of the three
distinct viral populations were also found".

That the "HIV DNA" may be "Even more divergent" than has been
generally accepted is best illustrated in a study published this year by
researchers from the United States. Because protease inhibitors are
becoming the drugs of choice for the treatment of "HIV infected"
individuals, and because "naturally occurring mutations in HIV-1
infected patients have important implications for therapy and the
outcome of clinical studies", these researchers performed a "sequence
analysis of the pr gene [protease gene] in 167 HIV-1 viral strains from
102 protease inhibitor naive patients collected from different
geographic regions of the United States". "Given the enzyme's relative
small size and the constraints in it structure imposed by function, it
was reasonable to conclude that sequence variability in HIV-1 would be
limited". To their surprise it was found that "A total of 41% of the
nucleotides and 49.5% (49/99) of the amino acids were variable. The
amino acid diversity seen in these USA viral isolates is much greater
than that previously reported for HIV-1 clade B viruses" and is also
greater than that seen in pr genes for all HIV-1 clades (40 out of 99,
40% of amino acids varying"!(170) At present, more so than in 1986 when
Gallo and colleagues reached their conclusion that "The rate of genetic
changes for the AIDS virus is more than a million fold greater than for
most DNA genomes and may even be tenfold greater than for some other RNA
viruses including certain retroviruses and influenza A virus", and in
1989, when the Pasteur researchers reached their conclusion that "the
task of defining HIV infection in molecular terms will be difficult",
there is no evidence which proves the existence of a unique molecular
entity "HIV RNA" ("HIV DNA"). In fact, there are a number of reasons why
the myriads of incommensurable "HIV DNAs" cannot be even described "in
terms of populations of closely related genomes, referred to as a
quasispecies".(153) These include: (a) Eigen and his colleagues
developed the quasispecies model to describe the distribution of
self-replicating RNAs. However, the "HIV RNA", is said not to be a self
replicating RNA, but replicates through a DNA intermediate; (b) the
self-replicating RNA of the RNA viruses appears to "demonstrate
remarkable stability in some situations. The type 3 Sabin poliovirus
vaccine differed from its neurovirulent progenitor at only 10 nucleotide
positions after 53 in vitro and 21 in vivo passages in monkey tissues.
In 1977, H1N1 influenza A virus reappeared in the human population after
27 years of dormancy with sequences mainly identical to those of the
1950s virus". Although Eigen's quasispecies model has been used to
describe the genome of RNA viruses, even 1% sequence differences in
these genomes are considered to represent "extreme variability". "Many
selective forces may stabilize virus populations. These stabilizing
factors may include the need for conservation of protein structure and
function, RNA secondary structure, glycosylation sites, and
phosphorylation sites. Even third-codon changes can be subject to
selective pressures. Recently, remarkable conservation of certain
protein domain sequences has been observed between completely unrelated
RNA viruses.(171) It is possible then to describe the "HIV DNA" even if
it has variation of 10% , not to mention 20 or 30 or 40% as is the case,
as a "population of closely related genomes, referred to as a
quasispecies"?; (c) Defining the concept of a quasispecies Eigen wrote:
"In the steady state that is eventually reached the best competitor,
designated the master sequence m, coexists with all mutant sequences
derived from it by erroneous copying. We designate this distribution of
sequences as quasispecies". However, to date, nobody has proven that:
(i) there is an "HIV" quasispecies which is ever in equilibrium; (ii)
the "closely related HIV genomes" are derived from a master sequence;
(iii) a master sequence has ever existed.

6.4.4 If the "HIV RNA" stretch is the genome of an exogenous
virus which infects individuals with AIDS or those at risk, then this
RNA (or cDNA) should be present in fresh uncultured tissue from all
these individuals and in nobody else. Furthermore, if in these
individuals there is massive HIV infection, as some of the best known
HIV experts claim,(172,173) Southern blot hybridisation should be more
than sufficient to detect it. The first such study was conducted by
Gallo and his colleagues in 1984. Using a Southern blot hybridisation
technique they tested many tissues from AIDS patients, including lymph
nodes. Summarising their finding they wrote, "We have previously been
able to isolate HTLV-III from peripheral blood or lymph node tissue from
most patients with AIDS or ARC" (they "isolated" it from approximately
50% of patients referred to by Gallo). "However, as shown herein,
HTLV-III DNA is usually not detected by standard Southern Blotting
hybridization of these same tissues and, when it is, the bands are often
faint...the lymph node enlargement commonly found in ARC and AIDS
patients cannot be due directly to the proliferation of
HTLV-III-infected cells...the absence of detectable HTLV-III sequences
in Kaposi's sarcoma tissue of AIDS patients suggests that this tumor is
not directly induced by infection of each tumor cell with HTLV-III...the
observation that HTLV-III sequences are found rarely, if at all, in
peripheral blood mononuclear cells, bone marrow, and spleen provides the
first direct evidence that these tissues are not heavily or widely
infected with HTLV-III in either AIDS or ARC".(96) These studies were
confirmed by many other researchers. The finding that when the results
were positive the hybridisation bands were "faint", "low signal" was
interpreted as proof that HIV seropositive individuals contain HIV DNA
in small numbers of cells and at low copy numbers, an interpretation
which became generally accepted, although Gallo and his colleagues had
an alternative explanation, "Theoretically, this low signal intensity
could also be explained by the presence of virus distantly homologous to
HTLV-III in these cells".(96) This alternative explanation has been
ignored by everybody, including Gallo. However, at a 1994 meeting held
in Washington sponsored by the US National Institute of Drug Abuse,
Gallo admitted "We have never found HIV DNA in the tumor cells of
KS...In fact we have never found HIV DNA in T-cells".174 Data which has
come to light since 1984 suggest that Gallo's and his colleagues'
alternative explanation may be a fact: (a) at present there is ample
evidence showing that normal human DNA contains sequences related to
HTLV-I and HTLV-II (see 6.3.2); (b) apparently, up until 1993, Gallo was
unaware of the existence of endogenous human retroviruses, (107) which
means that by "virus distantly homologous to HTLV-III" they could have
meant none other than the exogenous retroviruses Gallo claimed to have
discovered earlier, that is, HTLV-I and HTLV-II. However, at present
even Gallo admits that the human endogenous proviral sequences "comprise
about one percent of the human genome"; (c) some of the best known HIV
experts including Montagnier, Blattner and Gelderblom agree that the pol
and gag genes "may be highly conserved between subtypes of virus" (see
5.6). In a paper published in 1996 by Reinhart Kurth and his colleagues
one reads, "Retrotransposons evolved in a variety of organisms ranging
from protozoa to human beings. In these elements, RT genes are linked to
genes that code for polyproteins with the potential to self aggregate
and to form core particles. These proteins are the equivalents of the
retroviral capsid proteins usually designated group-specific antigens
(gag)...They [retrotransposons] may be either the derivative or
predecessors of retroviruses. Retroviruses differ from retrotransposons
by the presence of at least one additional coding region, the envelope
(env) gene".(175) In 1984, Gallo's group reported that the "HIV genome"
hybridised with the "structural genes (gag, pol, and env) of both HTLV-I
and HTLV-II.(56) Obviously, the finding of a positive hybridisation
"signal" at least with an "HIV" gag or pol probe is not proof for the
existence of the "HIV genome"; In fact, at present evidence also exists
which shows the presence of "HIV" sequences in non-infected tissues: (i)
although it is no longer accepted that HIV is transmitted by or is
present in insects, in 1986 researchers from the Pasteur Institute found
HIV DNA sequences in tsetse flies, black beetles and ant lions from
Zaire and the Central African Republic;(176) (ii) in 1985 Weiss and his
colleagues reported the isolation, from the mitogenically stimulated
T-cell cultures of two patients with common variable
hypogammaglobulinaemia, a retrovirus which "was clearly related to
HTLV-III/LAV"; Evidence included positive WB with AIDS sera and
hybridisation with HIV probes;(177) (iii) DNA extracted from thyroid
glands from patients with Grave's disease hybridises with "the entire
gag p24 coding region" of HIV;(178) (iv) In a study designed to address
the question whether the neuronal cells of patients with AIDS dementia
complex are infected with HIV, "the brains from 10 patients with AIDS
and neurological evidence of viral encephalitis and the brains from 5
patients without HIV-1 infection" were examined using an HIV gag probe.
"The antisense riboprobe hybridized to cells known to be infected with
HIV-1. It hybridised to HIV-1 infected A3.O1 cells as well as splenic
and renal lymphocytes obtained at autopsies from patients known to have
AIDS. The probe did not, however, hybridize to neurones in the brain
sections from 10 patients with AIDS...Surprisingly, when we applied the
control sense HIV-1 gag probe to the brain sections from patients with
AIDS, we observed specific hybridization to neuronal cells. Similarly,
when brain sections from five individuals not infected with HIV-1 were
examined, the HIV-1 sense probe detected transcripts in neuronal cells.
Our Northern blot analysis confirmed these results and demonstrated the
presence of a 9.0-kb polyadenylated transcript in brain tissues".179
Thus, either the positive hybridisation signals obtained with the
antisense probe are non-HIV-specific or, as the authors concluded, there
is a neurone-specific 9.0-kb transcript that shows extensive homology
with antisense gag HIV-1 sequences and that this transcript is expressed
in neuronal cells of both HIV-1- infected and noninfected individuals;
(v). Horowitz et al, "describe the first report of the presence of
nucleotide sequences related to HIV-1 in human, chimpanzee and Rhesus
monkey DNAs from normal uninfected individuals". They have "demonstrated
the presence of a complex family of HIV-1 related sequences" in the
above species, and concluded that "Further analysis of members of this
family will help determine whether such endogenous sequences contributed
to the evolution of HIV-1 via recombination events or whether these
elements either directly or through protein products, influence HIV
pathogenesis".(180) The inescapable conclusion therefore is that the
hybridisation studies do not prove that T-cells or any other cells of
AIDS patients and those at risk contain a unique molecular entity "HIV
DNA".

6.4.5 In the second half of the 1980s, in order to rescue the
concept of an "HIV genome", the HIV experts made extensive use of a
newly discovered process known as the polymerase chain reaction (PCR).
Although the PCR is a very useful tool in molecular biology there are
many problems associated with its use in studying the "HIV genome": (a)
The PCR is an extremely sensitive technique. Writing of his Nobel prize
winning discovery, Kary Mullis, himself rather ironically sceptical of
the HIV/AIDS hypothesis wrote, "Beginning with a single molecule PCR can
generate 100 billion similar molecules in an afternoon".(181) With such
amplification it is not difficult to detect even very low levels of the
"HIV genome". However, "a striking feature of the results obtained" by
1990 with PCR as with the standard Southern/Northern hybridisation, was
"the scarcity or apparent absence of viral DNA in a proportion of
patients".(182) In a further effort to rescue the "HIV genome", in the
1990s researchers from the Department of Genetics University of
Edinburgh introduced a modified version of PCR, the double PCR method or
nested PCR. "The double PCR overcomes the problem of limited
amplification of rare template sequences". They reported that, "Using a
double polymerase chain reaction which allows the detection of a single
molecule of provirus and a method of quantifying the provirus molecules,
we have measured provirus frequencies in infected individuals down to a
level of one molecule per 105 PBMCs...As a general rule, only a small
proportion of PBMC contain provirus (median value of samples from 12
patients one per 8.000 cells)"...samples from 7 of our 12 patients (60%)
contained one or more provirus per 104 cells...while samples from all
(100%) of our patients contained one or more provirus per 80.000 cells".
They concluded, "The most striking feature of the results is the
extremely low level of HIV provirus present in the circulating PBMC in
most cases".(182) There is no doubt that PCR can "amplify a DNA-needle
into a DNA- haystack" but even PCR cannot perform miracles.

In a review of Neville Hodgkinson's book, 'AIDS The failure of
Contemporary Science: How a Virus That Never Was Deceived the
World",(183), Sir John Maddox wrote, "the virus that never was has been
made more tangible" early in 1995 when "it became apparent that even in
the earliest stages of infection by HIV, the virus is far from
dormant".(184) Maddox is referring to two papers published in Nature in
1995. One by Ho et al where the authors claim to have shown that in
patients who have not received antiviral treatment the "plasma viral
levels ranged from...15 X 103 to 554 X 103 virions per ml";(172) the
other by Wei et al where it is claimed that the "plasma viral RNA levels
in the 22 subjects at baseline ranged from 104.6 to 107.2 molecules per
ml" and concluded that their study "suggests that virus expression per
se is directly involved in CD4+ cell destruction. The data do not
suggest an "innocent" bystander mechanism of cell killing whereby
uninfected or latently infected cells are indirectly targeted for
destruction by absorption of viral proteins or by autoimmune
reactivities".(173) These claims raise two obvious questions: (i) "The
majority of exogenous pyrogens are microorganisms, their products or
toxins", and "endogenous pyrogens are polypeptides produced by a large
variety of nucleated host cells including monocyte/macrophages" and
"lymphocytes, endothelial cells, hepatocytes, epithelial cells,
keratinocytes, and fibroblasts, as well as other cells...generally in
response to initiating stimuli triggered by infection or inflammation".
In addition, "many endogenous products result in the release of
endogenous pyrogens, thereby causing fever. Such endogenous substances
include antigen-antibody complexes, complexes with complement,
complement cleavage products, steroid hormone metabolites, bile acids
and some cytokines".(185) Since "the virus ["HIV"] is replicating 24
hours a day and from day one",(155) and "2X109 CD4+ cells [are] produced
and destroyed each day", and fever and "many of the associated features
of fever can be reproduced by infusions of purified cytokines, including
back pain, generalised myalgias, arthalgias, anorexia and
somnolence",(185) it is indeed surprising that such "massive" infection
and cellular destruction may remain largely, if not totally,
asymptomatic for prolonged periods of time in HIV seropositive
individuals; (ii) If there is such a "massive" HIV infection, why is it
not detected by standard hybridisation procedures and why, in order to
detect such "massive" infection, did not the authors use PCR which can
"amplify a DNA- needle into a DNA-haystack" or even nested PCR but were
obliged to determine "Viral RNA" with novel assays, "modified branched
DNA (bDNA) or RT-PCR assay and confirmed by QC-PCR" for which no details
are given?

One of the many problems (186,187) associated with the Ho and Wei
studies and the methods they employ is illustrated in a presentation at
the XIth International Conference on AIDS. Researchers from the Medical
School, Camden, New Jersy took a single plasma sample from a patient
"with a CD4 cell count of 123 cells/cmm" and divided it into ten
aliquots. The RNA from each sample was reverse transcribed and the cDNA
"was then amplified with an internal control DNA (mimic) using gag
primers...cDNA was also pooled from the initial 10 individual RT
reactions and QC-PCR was performed 10 times on pooled cDNA". They
reported that "The mean HIV-1 copy number for the 10 individual plasma
aliquots was 136,000 RNA copies/ml with a standard deviation of 76,9000
copies/ml (range 74,2000 copies/ml to 334,600 copies/ml). The mean HIV-1
copy number for the pooled cDNA assayed 10 times was 145,900 copies/ml
with a standard deviation of 61,900 copies/ml (range 84,500 copies/ml to
259,300 copies/ml)...the RT is not the source of variability in HIV-1
QC- PCR. Rather, variability is likely due to differences in
amplification of the target template and internal control used in the
QC-PCR assay".(188)

According to Maddox and Wain-Hobson both Ho and Wei and their
colleagues were able to reach their startling conclusions only after a
decade of HIV research because they teamed up with mathematicians and
because they were able to use "New techniques for assaying the low
levels of virus involved"! (italics ours). It is ironic then that
the strongest criticism of these studies have emanated from
mathematicians such as Frank Buianouckas from the Department of
Mathematics and Computer Science, City University, Bronx, New York USA
and Mark Craddock, School of Mathematics and Statistics, The University
of Sydney, Australia. "What is this viraemia of billions of RNA
particles that can only be seen with an undocumented branch-PCR or PCR
but not with a functional infectivity test?".(189) "My question is this.
Just what exactly will it take to get people doing HIV research to turn
away from high tech, unproven methods, arcane speculations about
molecular interactions etcetera etcetera and ask themselves 'do any of
us have the faintest idea what we are doing?'".(190) One can argue that
criticisms of the Ho and Wei papers by individuals from the HIV/AIDS
dissident movement is not to be unexpected but it is unheard of for one
group of HIV experts to criticise another as it happened with the Ho and
Wei studies.(191) In July 1995, as a result of "misgivings" about the
claims of Ho and Wei and their colleagues, "two dozen AIDS researchers
congregated in Berkeley, California...to challenge the establishment,
swap copies of their own manifestos, and enjoy the bonhomie of hanging
out for 2 days with fellow "alternative" thinkers", who concluded that
Ho et al and Wei et al "were short on compelling evidence that their
ideas were correct".(192) (b) According to researchers from the Walter
Reed Army Institute of Research, "the extensive use of the polymerase
chain reaction (PCR) to recover HIV-1 proviral DNA has favoured analysis
of the short amplicons that are most efficiently recovered by this
technique".(193) In fact, in the vast majority of cases the presence of
the "HIV genome" is proven by amplifying short "invariant regions" of a
"viral gene", usually of the gag gene. However, since it is accepted
that a significant proportion of the "HIV genomes" are defective,
finding a fragment of a gene is not proof of the existence of the whole
gene and even less so for the existence of the whole genome "HIV DNA" or
"HIV RNA", a point accepted by many HIV/AIDS researchers. (c) If a
unique molecular entity "HIV DNA" exists, then the same primers would be
able to amplify it, irrespective of where such unique DNA is found.
According to the same researchers, "Due to the extensive genetic
diversity of HIV-1, opportunities to identify a single primer pair
capable of amplification of diverse subtypes are limited".(193,194) In
fact, amplification results obtained with primers for different genes
from one subtype are not in complete agreement. For example, in the
first "HIV" PCR, two primer pairs to amplify the gag gene were used and
it was found that "some samples scored positive with only one of the two
primer pairs".(195) It is said that in the USA and Europe individuals
are almost exclusively infected with subtype B. Yet researchers from the
University of Edinburgh found that "The results obtained with the gag
and env primers were not in complete agreement. In 5 of the 28
replicates, either the gag or an env sequence was amplified but not
both".(182) A PCR study of 40 individuals using primers from the LTR,
gag and env regions was performed by French researchers including
researchers from the Pasteur Institute. Out of 38 positive samples, "34
were gag positive (90%) whereas env and LTR were detected in fewer cases
24 samples (63%) and 18 samples (47%) respectively...11 of 40 samples
were positive with three primer pairs, 16 with two primer pairs and 11
with only one primer pair".(196) Such discrepancies may be due to: (i) "
a false-positive reaction", which the authors themselves suggest but
which they say is unlikely; (ii) "the known genomic variability of HIV".
If this is the case then one cannot talk of the "HIV genome" as being a
unique molecular entity. Indeed, if such variability is entertained then
it may be only the lack of an immense variety of primer pairs that
prevents all of Homo sapiens from being "infected with HIV"; (iii) the
genome is defective. (d) No meaningful information can be obtained from
a test unless the test is standardised and it is shown to be
reproducible. No such data is currently available for the PCR. In fact,
since there are so many "HIV" subtypes and one has to use different
primers for different subtypes or even for the same subtype, it makes it
extremely unlikely that such data can ever be obtained. (e) By far the
most important parameter of a test is its specificity, that is, how
often a test is negative when the condition sought is absent. For PCR
one must have proof that the primers: (i) belong to a unique retrovirus
as defined in the procedures described in 6.1; (ii) the primer sequences
are found only in the unique retrovirus and nowhere else; No such
evidence exists for the "HIV" primers. In fact, since it is not possible
to say what the "HIV DNA" sequences are, it follows that it is also not
possible to be specific about what the primers represent. Even if one
assumes that the "HIV DNA" and thus the primers are specific to a
retrovirus since: (a) most of the "HIV" primers originate from the
leukaemic cell lines HUT78 (H9), CEM, and EBV-transformed cells; (b)
there is evidence that leukaemic cells and EBV- transformed cells
contain endogenous retroviruses, including the CEM cell line;88 (c)
"release of endogenous retroviruses can be induced by the methods used
to "isolate HIV"; (d) Gallo himself reported that the HUT78 (H9) cell
line "contained HTLV[-I] proviral sequences";(105) (e) no method exists
to separate one retrovirus from another; it is impossible to say that
the "HIV DNA" probes are HIV, or DNA probes of an endogenous retrovirus
or even an exogenous retrovirus HTLV-I; (iii) in a DNA (RNA) sample the
primers bind only to HIV sequences and not to any other non-HIV
homologous or non- homologous sequences. Again, no such data exists.
Furthermore, given the facts that: (a) "about one percent of the human
genome" consists of endogenous retroviral sequences; (b) homologies
exist between the genes of endogenous and exogenous retroviruses,
especially in the gag and pol genes, and between these genes and
cellular retroelements; specific binding of the "HIV" primers is most
unlikely.

Even if (i)-(iii) are proven one must still determine the specificity
of the PCR reaction, that is, show that no positive results are obtained
in individuals who are not infected with HIV. This can only be
determined by using HIV isolation as an independent gold standard, that
is, by comparing PCR with the procedures listed under (see 6.1). This
has not been done, a fact accepted by one of the best known HIV/AIDS
researchers, William Blattner "One difficulty in assaying the
specificity and sensitivity of human retroviruses [including HIV] is the
absence of a final 'gold standard'".(59) (f) At present some evidence
obtained without the use of a gold standard illustrates that the PCR
procedure is non-specific: (i) There has been only one study in which
the reproducibility, sensitivity and specificity of PCR were examined.
In this study, the gold standard used was not HIV isolation but
serological (HIV Western blot) status. In this investigation, Christine
Defer from the Laboratorie d'Ingenierie Moleculaire, Centre Regional de
Transfusion Sanguine including colleagues from the Pasteur Institute,
studied PCR testing proficiency in "Seven French laboratories with
extensive experience in PCR detection of HIV DNA". Four groups of
individuals were tested: those with "unequivocal HIV-positive test
results" (ELISA confirmed with Western blot); "individuals at low risk
of HIV infection who presented with a persistent and isolated anti-p24
antibody on Western blot"; "HIV-1 seronegative (on ELISA) individuals at
low risk of HIV infection (blood donors)", and "seronegative (on ELISA)
individuals at high risk of HIV infection (homosexual contacts of an
HIV-seropositive partner". From "two different peripheral blood
mononuclear cell panels...each consisting of 20 samples", the authors
compared PCR results in both seropositive and seronegative subjects. The
PCR was found to be non-reproducible, "False-positive and false-negative
results were observed in all laboratories (concordance with serology
ranged from 40 to 100%)", and "the number of positive PCR results did
not differ significantly between high- and low-risk seronegatives";(197)
(ii) The finding of positive PCR in eosinophils has been interpreted to
"suggest that eosinophils may act as host cells for HIV-1".(198)
However, "Formaldehyde-fixed eosinophils nonspecifically bind RNA probes
despite digestion with proteolytic enzymes and acetylation...When
preparations are treated with amounts of ribonuclease adequate to
destroy viral RNA, the eosinophilic binding remains";(199) (iii) One
group of researchers reported that "While evaluating a nested PCR
procedure for the detection of HIV, we found that primers for the env
gene of HIV-1 amplify human satellite DNA sequences in a small
proportion of blood donors to produce a fragment that is close in size
to the genuine HIV PCR fragment in ethidium-bromide-stained gels";(200)
(iv) Controls and even buffers and reagents may give positive HIV PCR
signals;(201) (v) Monocytes from HIV+ patients in which no HIV DNA can
be detected, even by PCR, become positive for HIV RNA after
cocultivation with normal ConA-activated T-cells";(202) (vi) it is
generally accepted that once infected with HIV, always infected.
However, a positive PCR reverts to negative when exposure to risk
factors is discontinued.(203)

In a study of 327 health care workers exposed by needlestick injuries
to the "human immunodeficiency virus", 4 had "one or more positive" PCR
tests. An additional 7 had "an indeterminate PCR test result on the
initial specimen". Later samples for all 11 were negative "none
seroconverted or developed p24 antigenemia" and "all of the subjects
remained healthy".(204,205) While the evidence for such occurrence in
adults is sporadic, it is much more often reported in children. However,
PCR is not used for routine diagnosis of HIV infection in adults and
rarely, if ever, is repeated. Unlike adults, PCR is very often used in
children, this being the case because "HIV diagnosis" is "complicated by
persistence of passively acquired maternal antibody". By 1995 numerous
studies in children (206-209) revealed the conversion of a positive PCR
to negative. One of the most recent reports was published in 1995 by
French researchers. In a six year cohort of 188 "infected" children
which was analysed retrospectively 12 (6.7%) "cleared HIV infection".
Each child had at least two positive PCR results at two separate time
points in the first year, followed by numerous (up to 7) negative PCR
results. For PCR the investigators used primer pairs for the gag, pol,
and env gene regions; and the test was considered positive "if at least
two genes were amplified". Commenting on their results the authors
wrote, "Three different rooms with separate air-conditioned circuits
were used for DNA extraction, PCR-buffer preparation, amplification and
blotting. Amplicons were never transferred in the area reserved for
unamplified sequences. Thus, positive PCR results are unlikely to be due
to contamination...Nevertheless, as our PCR assays are performed on
unmanipulated cells, culture contamination leading to false positive PCR
results is impossible...We therefore consider that the probability of
repeated contamination on successive samples from the same child is
scarce". The authors "could not find any correlation between either
neutralizing or antibody-dependent cellular cytotoxicity-mediating
antibodies and HIV clearance". Of 139 children born to HIV positive
mothers but who were "clearly negative", "eight were PCR-positive once
for a single viral gene (pol), three were positive twice for the pol
gene, and once of the three was also positive for the gag gene in a
single assay".(210)

In 1989, discussing their studies on human retroviruses, researchers
from the University of New York wrote, "Irrespective of the origin of
human retroviruses, their presence leads to both practical and
theoretical concerns. Presently, the major practical concern is that
effective use of PCR as a screening procedure for HTLV-I, HTLV-II and
HIV infections must always include appropriate controls to ensure that
no endogenous sequences contribute to positive signals. As previously
noted, HIV unique primers corresponding to the highly conserved reverse
transcriptase region shown in Fig. 1 function well in the PCR
amplification of HeLa DNA even at annealing temperatures around
60ø...Another practical concern is that the use of PCR for determining
the possible retroviral eitology of a variety of human diseases may be
complicated by endogenous retroviruses. Even if cDNAs are used for PCR
templates, the transcriptional activities of endogenous sequences must
be considered".(119) In an article published this year, where he
discusses the laboratory diagnosis of "HIV infection", Philip Mortimer
wrote, "Other diagnostic methods, e.g. p24 antigen testing, and proviral
DNA and RNA amplification exist, but these innovations in HIV diagnosis
need to be matched against the anti-HIV test and should be rejected
unless they fulfil a need that antibody testing fails to meet".(211)
According to researchers from the University of London, "The use of
polymerase chain reaction (PCR) for the diagnosis of HIV infection is
becoming more widespread and although not yet entirely reliable compared
with serology, has been of special value in HIV-seronegative intravenous
drug users".(200) If PCR needs to be matched against the "HIV" antibody
test because it is less reliable than serology then given the fact that
at present there is no evidence which shows that a positive "HIV"
antibody test is proof of HIV infection, (89) one has no choice but to
agree with Shoebridge et al that "until further molecular and biological
studies are carried out, it will be unsure as to what detection of HIV-1
DNA, even when shown to be HIV-1 really means.(212) In analysing the
"HIV" molecular biology one cannot help reflecting on the words of Sir
John Maddox, "Is there a danger, in molecular biology, that the
accumulation of data will get so far ahead of its assimilation into a
conceptual framework that the data will eventually prove an encumbrance?
Part of the trouble is that excitement of the chase leaves little time
for reflection. And there are grants for producing data, but hardly any
for standing back in contemplation".(213)

CONCLUSION

The present data do not prove the existence of a unique molecular
entity "HIV DNA" which constitutes the genome of a unique, externally
acquired retrovirus, HIV. Neither is there any proof for the existence
of an "HIV quasispecies". Nor is it possible to say what exactly the
different "HIV DNAs", the probes and primers derived from these DNAs and
the sequences in the cellular DNA with which they hybridise
represent.

7.1 Before the cited evidence is discussed in detail, to avoid
misunderstanding, it will be helpful to define some terms including
cloning of DNA, transfection and virus cloning, as well as the evidence
that must be presented to claim proof of these phenomena:

Plasmid- freely replicating, circular chromosomal elements present in
bacteria. They duplicate independently of the main chromosomal element
and are frequently used to "carry" a DNA fragment into a cell.

DNA cloning- the production of identical copies of a DNA fragment,
any DNA fragment, from an ancestral DNA fragment by splicing it into a
suitable cloning vehicle, for example, a bacteriophage or plasmid;

Transfection- the introduction of exogenous DNA into cells and its
ability to replicate and express itself in these cells, that is,
transcription of DNA into RNA, translation of RNA into proteins. The
genetic material does not have to be of viral origin and transfection
can be achieved by various methods. As far back as 1969 it was known
that these methods may include "infection of cells with bacteria and
viruses, formation of hybrids of two cell types by fusion,
transplantation of isolated single nuclei in eggs and embryos,
microinjection of nuclei and mitochondria fractions, and pinocytic
uptake of purified DNA". In that year Margit Nass from the University of
Pennsylvania, taking advantage "of the phagocytic properties of mouse
fibroblasts (L cells) grown in suspension culture" demonstrated that,
"Mouse fibroblasts (L cells) in suspension culture incorporated isolated
chloroplasts of spinach and African violets and isolated mitochondria of
chicken liver...Green cells divided like normal cells. Green
chloroplasts were followed for five cell generations or 5 days, at which
time hybrid cells were greatly outnumbered by nongreen progeny cells".
(214) By 1989 it was realised that the delivery of DNA into cells could
be facilitated by polycationic reagents such as poly-DEAE dextron and
polyornithine. "An aliquot of the aqueous reagent is simply added to the
tissue culture experiment together with the DNA or RNA of
interest".(215) (It is of interest that cultures/cocultures derived from
tissues of HIV positive and AIDS patients are treated with the
polycation polybrene and/or oxidising agents which may lead to the
formation of cations). In 1990, researchers from the University of
Wisconsin showed "that injection of pure RNA or DNA directly into mouse
skeletal muscle results in significant expression of reporter genes
within muscle cells...RNA and DNA expression vectors containing genes
for chloramphenicol acetyltransferase, luciferase, and á- galactosidase
were separately injected into mouse skeletal muscle in vivo. Protein
expression was readily detected in all cases, and no special delivery
system was required for these effects. The extent of expression from
both the RNA and DNA constructs was comparable to that obtained from
fibroblasts transfected in vitro under optimal conditions".(216) One
year later another group of researchers from the USA showed that after
direct injection into animal hearts "of the firefly luciferose gene
coupled to the myosin heavy chain...the heart can be transfected in vivo
with greater efficiency than the skeletal muscle".(217)

Virus cloning-the introduction into cells of genetic material, DNA or
RNA which has been proven beforehand to be the genome of a virus
followed by the appearance in the same cells of viruses identical in
every aspect to the viruses from which the genomic material originated.
Before one can claim proof of cloning of a retrovirus one must: (a)
Obtain a particle(s) separated from everything else (isolated) and show
that the particle contains, among other molecules, proteins and nucleic
acids (RNA), and that the particle(s) is indeed an infectious particle
(see 6.1); (b) Show that there is a direct relationship between the
particles' nucleic acids and proteins, that is, the proteins are coded
by the nucleic acids (the viral genome); (c) Introduce the viral genome
(RNA or DNA) into cells and show that the DNA (cDNA) is integrated into
the cellular DNA and is transcribed into RNA and the RNA is translated
into proteins (transfect the cells); (d) Show that the cells produce
particles and that the particles' proteins are coded by the particles'
nucleic acids; (e) Show that the particles' nucleic acids and proteins
are identical with those of the ancestral particle and that they too are
viral particles; (f) Because all cells contain retroviral genomes, which
under appropriate circumstances may be expressed in culture, that is,
both the cells in the culture from which the original particles were
obtained as well as the transfected cells may release identical
retroviral particles even if there is no cloning, when one attempts to
clone a retrovirus a control culture is of quintessential significance.
The only difference between the control and the cells transfected with
the viral genome should be that in the control cultures one should use
some other genes for transfection. This is because, under suitable
culture conditions, the very act of transfection may result in
retroviral expression including the production of retroviral particles.
It is obvious that retrovirus cloning is not synonymous with retrovirus
isolation, in fact, for cloning one must isolate the virus twice, the
first time to obtain the viral genome and the second time to prove that
the particles, if any, released by the cell after introduction of the
viral genome, are identical with those from which the genome was
originally obtained.

7.2 In 1985 Fisher, Gallo and their colleagues published an
article entitled, "A molecular clone of HTLV-III with biological
activity".(94) "The phage clone ^HXB-2 [see 6.2.2] which contains
full-length provirus (~10 kilobases, kb) with cellular flanking
sequences (12.7 kb total length)" was inserted into the plasmid pSP62.
"Similarly, a 13.7 kb Eco RI fragment of ^CH-1 (a molecular clone
containing ~9.0 kb of HTLV-I proviral sequences) was inserted into"
another plasmid, pSV2gpt. "These plasmid constructs [pHXB-2D, pCH-1gpt]
were then transfected into DH-1 bacteria and used in protoplast fusion
experiments". pCH-1gpt and yet another plasmid containing "no HTLV
sequences (pSVneo)" were used as controls. (No reasons are given why
they used three different plasmids). PHA stimulated cord blood
mononuclear cells "were then fused with bacterial protoplasts carrying
"the plasmids". "Three parallel fusions using cells from different
individuals were established for each plasmid". (It is not clear if they
used cells from 3 or 9 individuals, if the latter, this is an additional
reason why the cloning conditions could not have been identical).

(a) Spent medium "was concentrated 10-fold and assayed for the
presence of reverse transcriptase" using A(n).dT(15), at days 5, 11, 14
and 18 after fusion. If the conditions used for transfection were
identical and if transcription indicated the presence of a retrovirus,
then one would expect RT to be present in the cultures with pHXB-2D and
the three cultures with pCH-1gpt. However, DNA synthesising activity was
reported only in two cultures with pHXB- 2D, (the activity in one of
them was less than half the other at each sampling point), and no
mention is made regarding the activity in the third culture.
Furthermore, for some unknown reason, the DNA synthesising activity was
reported only for 18 days after transfection when it was said to be
maximum. Unlike RT activity, the viability of the cells in the cultures
was determined repeatedly starting before transfection and up to 32 days
afterwards. The results were reported as the mean of the three cultures
for each plasmid. If the viability of the cells was determined by the
expression of retrovirus present in the cultures and if HIV and HTLV-I
possesses the biological properties attributed to them, then one would
expect that the number of cells in the cultures containing pSV2neo to
remain constant, in the cultures containing pHXB-2D to decrease, and in
the cultures with pCH-1gpt to increase. They reported that between day
18 and 32 the number of viable cells decreased in all cultures. The
decrease was most pronounced in the culture with the "HIV clone", and
appeared earlier, "By day 18, however, the number of viable cells in
cultures transfected with pHXB-2D has fallen dramatically". In other
words, the highest cell death occurred before maximum HIV (RT)
production and even before the full "HIV DNA" was integrated into the
cellular DNA (see below). Furthermore, since apparently no RT activity
was detected in one of the three cultures with pHXB-2D, in this culture
the cell number should have remained constant.

(b) Results of the hybridisation studies are given only for pHXB- 2D,
and even there for only one of the three cultures with this plasmid.
"The presence of HTLV-III sequences was demonstrated by Southern blot
analysis" using "insert" from the molecular clone ^BH-10, "an incomplete
viral clone of HTLV-III". "A 10-kb band, corresponding to unintegrated
linear virus, was detected in undigested DNA samples prepared 14 days
after transfection. Digestion with XbaI revealed three distinct band at
11, 10 and 5.2 kb...these bands probably represent the nicked circular,
linear and closed circular forms of unintegrated HTLV-III
respectively...Digestion with HindIII, an enzyme which cuts the HTLV-III
genome of pHXB-2D six times, yielded bands at 4.5, 2.0 (doublet), 1.7
and 0.6 (a doublet)...This restriction pattern is clearly different from
that of H9/HTLV-IIIB...High relative molecular mass 'smears' were not
observed when DNA was digested with BamHI. Therefore, we have no direct
evidence that transfected HTLV-III DNA is integrated in the host cell
genome...In time-course experiments (Fig. 36), DNA isolated from a
single culture 6, 11, 14, 18 and 31 days after transfection with
pHXB-2D, was digested with BamHI and analysed for HTLV-III sequences.
Six days after transfection an 8.6 kb DNA fragment was detected as a
faint band; 18 days after transfection it was possible to detect a 1.5
kb DNA fragment in addition to the 8.6 kb fragment...No HTLV-III
sequences were detected 31 days after transfection". Despite these
findings, the time-course experiments were interpreted "as evidence that
cells originally transfected with pHXB-2D are able to produce fully
infectious virus which is then transmitted within the culture"!

(c) The pHXB-2D transfected umbilical cord lymphocytes were reacted
with "monoclonal antibodies against the HTLV-III-gag-related proteins
p24 and p15...maximum expression was observed 15 days after
transfection, when 4-11% and 5-9% of cells were reactive with antibody
to p15 and p24, respectively (data not shown)...In comparison, among
H9/HTLV-III cultures, a much larger proportion of cells (70-90%) was
positive for p24 and p15". In addition to the many problems associated
with the interpretation of a positive antibody/antigen reaction,
especially with umbilical cord cells and the gag antigens (antibodies),
as proving HIV infection, it is also interesting to note that: (i)
maximum antibody/antigen reactions preceded maximum reported RT activity
and hybridisation bands; (ii) No mention is made regarding the antibody
reactivity with the pSV2-neo transfected cells but "cord blood cells
removed 18 days after transfection with pCH-1gpt (HTLV-I clone) were not
labelled by these antibodies". However, if as Gallo claims: (a) the gag
genes of HIV and HTLV-I are homologous; (b) there is cross-reactivity
between the p24 proteins of the HTLV-I and HIV-1; the reported finding
that the "monoclonal antibodies against the HTLV-III gag-related
proteins" did not react with the pCH-1gpt transfected cells is
inexplicable. Their immunological findings led them to write, "The
finding that, at any stage, only a minor population of the transfected
cells are apparently infected by the virus (<15% express viral
proteins) suggests that the cytopathic effects may not result solely
from direct viral infection". However, if the dramatic fall of viable
cells in the pHXB-2D transfected cultures where only a minority of cells
are "infected" is caused either directly or indirectly by "the clone of
HTLV-III with biological activity" (cytopathic effects), why are such
effects not also observed in the H9/HTLV-III cell line where a much
higher percent of cells is "infected" but such cells divide
indefinitely? Especially when one considers the fact that the H9 (HUT78)
cell line originates from a patient who "had malignancies of mature T4
cells"6 and HIV is said to specifically destroy the T4 cells.

(d) Fisher and colleagues published an electron micrograph showing
extracellular but not budding, virus-like particles some of which had a
diameter of 100nM. However, they did not prove that the particles were
viral particles or even that they had any of the other morphological and
physical characteristics of retroviral particles.

7.3 In 1986 Levy and his colleagues published a paper entitled
"AIDS retrovirus (ARV-2) clone replicates in transfected human and
animal fibroblasts".(218) The molecular clone ^9-B of ARV-2 (see 6.2.3)
was inserted into the plasmid pSp65. The p9B-7 thus obtained and ^9B-7
were used to transfect the human monocytic cell line U937 as were the
Jurkat and HUT-78 cell lines. ARV was detected by the presence of "RT
activity in the culture supernatant...ARV production was detected in the
Jurkat and U937 cells at 36 to 44 days after transfection by the
presence of reverse transcriptase (RT) activity...Virus replication was
detected at 5 days in the HUT-78 line, with RT activity reaching over
200.000 cpm/ml...Virus from each culture was subsequently passed to
mitogen stimulated normal human peripheral mononuclear cells
(PMC)...Reverse transcriptase activity increased to over 106 cpm/ml
within 14 days after the virus from the HUT-78 cells was passed to fresh
human PMC". The NIH 3T3 (mouse), MIL (mink lung), COS-7 (African Green
monkey), and RD-4 rhabdomyosarcoma (human) cells were also transfected.
In all cells RT activity was detected within 5 to 14 days after
transfection. "The detection of virus was enhanced by cocultivation of
the fibroblast cells with mitogen-stimulated normal human PMC...added
every 3 to 6 days". Protein extracts of "PMC infected with virus
recovered from transfected MIL cells", COS-7 cells and HUT-78 were
electrophoresed and reacted with "serum positive for antibodies to
ARV...Extracts of the infected HUT-78 cells and PMC contained all the
antigens of ARV as demonstrated by immunoblotting (Fig. 2). These
included the envelope proteins gp160, gp120, gp41, and the gag proteins
of molecular weight 55K, 25K, and 16K". No such reactions were reported
with the "non-infected" PMC. However, even Montagnier reported that at
least one protein, gp41 from non-infected cells react with patient sera.
The difference may be due to the fact that apparently Montagnier
stimulated the non-infected cells but Levy did not. Again, while in
normal non-stimulated cells patient sera do not react with a p16-18
protein, the same proteins are detected in normal, non-infected but
stimulated cells.(219-222) Levy and his colleagues also found that "The
virus recovered from all the cells was cytopathic for HUT-78 cells...The
virus produced in HUT-78 cells showed cytopathic effects (fusion,
balloon degeneration) typical of AIDS retroviruses". If the cytopathic
effects are caused by a virus which appeared as a result of cloning then
Levy et al managed to prove an effect of HIV on HUT-78 (H9) which to
date nobody else has managed to demonstrate. (It is true that in 1986
nobody apart from Gallo and his colleagues knew that HUT78 is actually
HT (H9)).

7.4 In 1993 Barnett, Levy and their colleagues published a
paper entitled "Distinguishing features of an infectious molecular clone
of the highly divergent and noncytopathic human immunodeficiency virus
type 2 UC1 strain". This study by Barnett, Levy et al refers to HIV-2.
Since HIV-2 is said to be totally different from HIV-1, its isolation or
cloning, even if true, in not proof for the isolation or cloning of
HIV-1. Nevertheless, since it has been cited a few comments may be
worthwhile. The "molecularly cloned virus (HIV-2UC1mc or UC1mc" was
obtained as follows: The cellular DNA of "UC1-infected SupT1 cells", was
"subjected to partial digestion with EcoRI. The digestion products were
size fractionated on NaCl gradients and then ligated to EcoRI-digested
EMBL4. Plaques were screened by hybridization to a mixture of DNA probes
including simian immunodeficiency virus from macaque, HIV-2ROD env cDNA
clone E2, and an HIV-1SF2 preparation enriched for gag-pol
sequences...Approximately 2 million plaques were screened, and 12
positive plaques were obtained following successive rounds of plaque
purification and hybridization. Of these 12 positive clones, only 1 was
found to contain full-length HIV-2 proviral DNA following restriction
enzyme analyses. Lambda-cloned UC1mc was transfected into RD cells by
calcium phosphate precipitation, and infectious virus was recovered
following cocultivation of these cells with
phytohemagglutinin-stimulated normal PBMC" and this "virus" was used to
transfer to other cell lines. Proof for virus cloning and the existence
of "infectious virus" was obtained as follows: "Culture supernatants
were assayed every 3 or 4 days for reverse transcriptase activity. Cell
samples were also tested for viral protein expression by an indirect
immunofluorescence assay. Cultures were examined at 2- or 3-day
intervals by light microscopy for cytopathic effects such as the
appearance of syncytia, large cells, ballooning cells, and cell debris.
Cell viability counts were determined by trypan blue dye exclusion.
Immunoblot analyses were performed as described previously by using
virus lysates prepared from cell culture supernatants of virus-infected
Molt4/8 cells. The sera came from HIV-infected individuals or from a
rabbit immunized with recombinant HIV-2ST gp120". They reported, "UC1mc
grew well in the Supt1, Molt4/8, and HUT78 T-cell lines but did not
exhibit productive infection of Jurkat or CEM cells...UC1mc demonstrated
relative inability to induce syncytium formation, kill cells, and
down-modulate surface CD4 expression in infected cells [does Levy and
his colleagues now agree with us80 that the apparent loss of CD4 cells
is not due to their destruction by "HIV", but to the ability of the
cultures to "down-modulate surface CD4 expression"?]...The molecular
sizes of the UC1mc viral proteins and their reactivities with various
sera were determined by immunoblot analysis. While most of the UC1 and
UC1mc viral proteins were reactive with sera from HIV-2 infected
individuals, the cell surface Env glycoprotein (gp140: SU) was usually
poorly reactive with these sera compared with the gp140s of other HIV-2
strains (e.g., HIV-2UC3) shown). In contrast, the UC1mc and UC1 gp140
molecules appeared to react well with Env-specific rabbit antiserum
raised against recombinant HIV-2STSU protein". For the molecular
characterisation of UC1mc, "The entire UC1mc genome was subjected to DNA
sequence analysis to determine its genetic structure and the relatedness
of its deduced proteins structure to those of other known HIV strains.
The proviral DNA sequence of UC1mc was found to be 10,271 bp long, and
its overall genetic structure appeared to be similar to that of other
sequenced HIV-2 strains...By sequence analysis, UC1mc appeared to
diverge substantially from most other HIV-2 strains. The differences
were most noticeable in the very low percentages of identify of
the amino acids sequences of Env; viral regulatory proteins Tat, Rev,
and Nef; and viral accessory proteins Vif, Vpx and Vpr. The divergence
of UC1mc was more subtle but nevertheless significant in the generally
more conserved Gag and Pol proteins"(223) (italics ours).

7.5 COMMENTS

Neither Fisher et al, Levy et al nor Barnett et al satisfied the
conditions absolutely necessary to claim cloning of a retrovirus, HIV.
Nor was it possible for them to so do. To molecularly clone a retrovirus
first one must obtain the retroviral RNA and this can only be obtained
by isolating the retrovirus. NO ISOLATION NO CLONING. However, to
date not only has no researcher isolated a unique retrovirus from fresh
tissues of AIDS patients or even from cultures/cocultures containing
material from these patients but neither has any researcher proven the
existence of particles, viral or non-viral, which satisfy the principal
morphological and physical properties of retroviruses.(146) Fisher et
al, Levy et al and colleagues, by various means, but with no proof that
it belonged to a particle, any particle, selected fragments of DNA, no
two of which were the same either in composition or length and called it
"HIV DNA" (see 6.2). Subsequently, they attempted to introduce the "HIV
DNA" into cells using well known techniques by which its is possible to
introduce any DNA, viral or non-viral, into cells. Irrespective of what
is meant by "HIV DNA", given the techniques they used, it is highly
probable that they succeeded. However, proof can only be claimed by
sequencing "HIV DNA" both before and after cloning into the cells and
none of these groups did so. The only evidence presented by the above
workers to this effect and indeed to virus cloning was: (a) The
detection in cell cultures of RT activity (transcription of A(n).dT15);
(b) The finding in cells of proteins ("the envelope proteins gp160,
gp120, and gp41, and gag proteins of molecular weight 55K, 25K and 16K")
which react with antibodies to p24 and/or with sera from AIDS patients.
However, thus far, nobody has proven that any of the above proteins
which are present in cell extracts and which may react with AIDS patient
sera are actually coded by the "HIV" env and gag open reading frames
(see 5). Neither are the presence of viral-like particles in the culture
supernatants nor transcription of A(n).dT15 proof for the existence of
HIV or of any retrovirus endogenous or exogenous (see 3.0). Even if
there was proof that the particles were actually retroviral and that
reverse transcription of A(n).dT15 was induced by a retroviral enzyme,
the proteins were retroviral proteins and the antibodies were
specifically directed against such proteins, their finding in cell
cultures is not proof of transfection of "HIV DNA" and even less of
"HIV" cloning. All of these phenomena may be caused by an endogenous
retrovirus, especially if one considers the type of cells used,
leukaemic and umbilical cord lymphocytes, and the conditions, chemical
stimulation and co-culture techniques. According to Kurth and his
colleagues, "indirect evidence has accumulated over the past years that
some endogenous proviral loci must also be expressed in
humans...Expression of retroviral information was also suggested by the
demonstration of reverse transcriptase activity and by the detection of
antigens cross-reactive with animal retroviral antigens in a variety of
human cells and tissues".(116) AIDS patients' sera contain antibodies
directed against many self and non-self antigens including lymphocytes
(89,224,225) and sera from 70% of AIDS patients react with antigens of
"The viruses in all of us", that is, endogenous retroviruses.(175) In a
1989 publication by researchers from Sweden, Japan and the USA one
reads: "In the 1960s and 1970s new techniques (morphological,
immunological, and molecular biological) became available...not only to
find exogenous or endogenous retroviruses, but also to correlate
retrovirus expression with certain human diseases...Electron microscopic
studies revealed particles with a retroviral morphology in several
normal and neoplastic human tissues and also in milk, urine and several
other effusions. Sensitive radioimmunoassays were developed which led to
the detection of antigens [including gag proteins in umbilical cord
blood sera] related to the proteins of known exogenous murine and
primate retroviruses and reverse transcriptase (RT) was found in
different normal and neoplastic tissues".(108) "Three HERV-R [human
endogenous retrovirus-R] polyadenylated mRNAs (9, 7.3 and 3.5 kilobases)
are expressed in first trimester and term placentae villi. A
comprehensive survey of HERV-R expression in human tissues revealed that
most other tissues also express the 9- and 3.5-kilobase mRNAs at a level
of about 10% of that in the placenta...The greatest expression besides
the placental villi was in the monocytic leukemia cell line U937", one
of the cell lines employed by Levy et al. Another of the cell lines used
by Levy et al in the 1986 study, COS-7, was from an African Green
monkey. Since then it has been shown that African Green monkeys are
infected with SIV and even earlier, 1983 they were said to be infected
with "adult T-cell leukemia virus".(226) The RD cell line used by Levy
is a human rhabdomyosarcoma cell line and for many years these cells
have been known to express viral information and to release
retroviral-like particles.(227) For cloning, Fisher et al and Levy et al
obtained their "HIV DNA" from the HUT78 (H9) cell line. This is also the
cell line from which Fisher and colleagues obtained most for their
evidence for "HIV-1 cloning". Even if one assumes that the "HIV DNA" is
indeed retroviral, for which there is no proof, it cannot be assumed to
be the "genome of HIV". According to Gallo the HUT78 (H9) cell line is
infected with HTLV-I.6 If so, then all HUT78 cell cultures, and the
clones derived from it, "infected with HTLV-III" or non-infected, and
the material from these cultures which bands at 1.16 gm/ml, should
contain HTLV-I, and thus RT and retroviral particles. Furthermore,
because about 25% of AIDS patients have antibodies to HTVL-I, and the
immunogenic proteins of HTLV-I and HIV have the same molecular weights,
then approximately 25% of the non-infected HUT78 (H9) cultures in
addition to RT and particles, should have, in the Western blot, the same
bands as those of the "HTLV-III infected" cultures. Thus, the cell
extracts from the HUT78 cells and the Western blots will erroneously
appear positive for HTLV-III. Both Gallo's and Montagnier's groups
showed that the gag and pol genes of HTLV-I and HIV-1 are homologous.
This means that the HUT78 cell line should have "HIV DNA" sequences even
when not transfected with "HIV DNA".

Unlike Fisher et al, Levy et al did not perform hybridisation
studies. However, Fisher, Gallo and their colleagues could not find
evidence that the "HTLV-III DNA is integrated into the host cell
genome", a step absolutely necessary in cloning and production of
retroviruses. Nor has anyone of these researchers shown that the DNA is
transcribed into RNA. For transfection, in addition to proving
integration of the "HIV DNA" into the host cell genome and its
transcription into RNA, one must also prove that the RNA is translated
into proteins.

CONCLUSION

To claim that "The existence of the retrovirus HIV predicts that HIV
DNA can be isolated from the chromosomal DNA of infected cells", one
must first have proof of the existence of a unique molecule of DNA which
is the genome of a unique retrovirus particle, HIV-1, which can only be
obtained by isolating the retroviral particle. At present there is no
such proof. Fisher et al and Levy et al selected a portion of the RNA
which from the supernatant of "infected" HUT78 cells banded at 1.16gm/ml
or had a certain length, reverse transcribed it and called it "HIV-1
DNA" (see 6.2.2; 6.2.3). However, since neither they nor anybody else
before or after them has shown that this RNA (cDNA) was even the
constituent part of a particle, any particle retroviral or otherwise,
the claim that the DNA is "Full length HIV-1" or "HIV- specific" cannot
be substantiated. In the cell extracts of "transfected" cells Fisher et
al and Levy et al found some proteins with molecular weights similar to
the "HIV proteins" which reacted with AIDS patient sera. They also found
reverse transcription of A(n).dT15 in the cell supernatant but presented
no evidence that the proteins or the RT were constituents of a particle,
viral or otherwise, and thus cannot claim that they have proven that the
"transfected" cells "produce particles that contain reverse
transcriptase, HIV specific antigens". Although Fisher and colleagues
had an electron micrograph showing virus-like particles in the culture
supernatant, they did not prove that the particles were indeed
retroviral particles, or even that they had some of the most basic
morphological and physical features of retroviral particles and thus
they "could reflect non-viral material altogether". Fisher et al, Levy
et al and Barnett et al did not start with RNA (cDNA) proven to be the
RNA of a retrovirus and did not obtain retroviral particles proven to
contain the same RNA, a most basic requirement for cloning. In fact,
given their evidence they cannot even claim transfection of cells with a
DNA, viral or non-viral.

8. "IDENTIFICATION OF HIV"

8.1 "The existence of HIV predicts that infected cells contain a
unique, virus specific DNA of 9150 nucleotides that cannot be detected
in DNA of uninfected cells".

The genome of a retrovirus cannot be identified on the basis of the
length of a RNA (cDNA) fragment and its presence in some but not other
cells.

8.1.1 Using fragments of "HIV DNA" as hybridisation probes or
primers, positive results with both standard hybridisation and PCR have
been obtained from DNA of "uninfected" human cells and insects (see
6.4.4). It is a fact that: (a) hybridisation of nucleic acids of
exogenous retroviruses "from different species gives a pattern which is
the same as the phylogenic relatedness among their natural hosts",(228)
a relationship which led retrovirologists including Gallo to conclude
that exogenous retroviruses "are derived from cell genes"; (b) The
existence of endogenous human retroviruses has been proven using
hybridisation probes derived from endogenous and exogenous animal
retroviruses. If this is the case and if "HIV DNA" is the genome of an
exogenous human retrovirus, the non-infected human genome should contain
sequences which will hybridise with "HIV DNA" probes. There can be two
reasons why such findings have not been reported more often: (i) Most
HIV researchers ignore one of the most fundamental requirement of basic
experimental research, that is, controls. In the rare instances where
controls are used, they are not suitable (see 6.1). In the 1970s, Gallo,
Gillepsie and their colleagues were saying that the success of the
"hybridization assay appears to depend on the biological history of the
virus", and on the physiological state of the cells.(125,228) In a large
study published in 1975 entitled "Relationship between Components in
Primate RNA Tumor Viruses and in the Cytoplasm of Human Leukemia Cells:
Implications to Leukemogenesis", the aim was to show that human leukemia
cells but not normal cells have properties associated with retroviruses
including retroviral genomic sequences. It was reported that "The human
leukemic blood cell cytoplasmic particle that contains reverse
transcriptase activity is capable of synthesizing DNA in vitro, using
endogenous RNA as both template and primer. This endogenous activity has
been used to learn about the nature of the particle itself. Many
intracellular cytoplasmic particles or organelles (described generally
in Table 8) can carry out endogenous DNA synthesis in vitro. These
include mitochondria, small cytoplasmic particles of low density,
1.10-1.16 g/cc in sucrose density gradients, and small cytoplasmic
particles of higher density, 1.17-1.19 g/cc in sucrose density
gradients...Small particles have been detected in the cytoplasmic
fraction of phytohemagglutinin-stimulated lymphocytes from normal
donors...These particles carried out endogenous DNA synthesis, and the
resulting DNA population contained sequences related to genomes of RNA
tumor viruses...Viral-related sequences were found in patients with
several types of leukemia, including AML, CML, CML-A and CLL...Attempts
to detect viral sequences in RNA of leukemic cells by hybridizing DNA
synthesized by animal viruses to RNA isolated from cytoplasmic small
particles (the reciprocal hybridization experiment) in our hands fails
to find differences in sequences in RNA of leukemic and dividing normal
[PHA stimulated] human peripheral white blood cells. It has been
reported by others that radioactive DNA probes synthesized by MuLVR
hybridize to cytoplasmic RNA from leukemic, but not normal white blood
cells. A difference between our experiments and those previously
reported is that the normal human cells used as a source of RNA are
actively dividing while most of those used in previous studies were
not"(125) (italics ours); (ii) The "HIV RNA" is not the genome of
either an exogenous or an endogenous retrovirus or even the transcribed
DNA fragment present in un-"shocked" cells.

8.1.2 Most of the positive results in "uninfected cells" have
been found by using probes and primers for one or at most two genes or
even gene fragments. The "great majority" of HIV studies, encompass "2%
to 30% of the genome".(163) However, finding fragment of a gene or even
a gene is not proof for the existence of the HIV genome.

8.1.3 Montagnier and his colleagues reported the "HIV DNA" to
be 9 ñ 1.5 Kb91 whereas Gallo and his colleagues reported that "The
overall length of the HTLV-III provirus is approximately 10
kilobases".(96) In Levy and colleagues' first study of the "HIV genome",
the "broad band (>15 Kb) represents provirus integrated into host
cell DNA".(98) In 1995, Pasteur researchers reported that "The complete
9193-nucleotide sequence of the probable causative agent of AIDS,
lymphadenopathy-associated virus (LAV), has been determined. The deduced
genetic structure is unique; it shows, in addition to the retroviral
gag, pol, and env genes, two novel open reading frames we call Q and
F".(229) In the same year, Gallo and his colleagues reported their
results on the "HIV" nucleotide sequences using clone BH10 but also
added, "The sequence of the remaining 182 bp of the HTLV-III provirus
not present in clone BH10 (including a portion of R, V5, tRNA primer
binding site and a portion of the header sequence) was derived from
clone HXB2...Of note is the presence of a fifth open reading frame
(nucleotides 8, 344-8991) designated 3' orf, present in clone BH8 but
truncated in BH10". They concluded, "The complete nucleotide sequence of
two human T- cell leukaemia type III (HTLV-III) proviral DNAs each have
four long open reading frames, the first two corresponding to the gag
and pol genes. The fourth open reading frames encodes two functional
polypeptides, a large precursor of the major envelope glycoprotein and a
smaller protein derived from the 3' terminus long open reading frame
analogous to the long open reading frame (lor) product of HTLV-I and
-II...The HTLV-III provirus is 9,749 base pairs (bp) long".(32) In 1990
the HIV genome was said to consist of ten genes.230 This year Montagnier
reported that HIV possesses eight genes (7) and Barr‚-Sinoussi,(8) HIV
has nine genes.

To date, no two "HIV DNA" of the same length have been reported and
moreover, it is accepted that most "HIV genomes" are defective. Even if
all the genes can be amplified by PCR, it still does not mean that the
"full-length HIV genome" is present. For example, in 1995 the nef gene
of 3 of the blood recipient members of the Sydney "Bloodbank" cohort and
of the donor were amplified by PCR. "The resulting amplified fragments
for the 3 recipients ranged from 410 bp to 680 bp. One recipient yielded
fragments of two sizes...The amplified fragment from the donor (D36) was
~ 550 bp in length, indicating a deletion of ~290 bp...compared with
~840-bp fragment from the molecular clone pNL4-3".(231) In 1995 David Ho
and his colleagues "analyzed by polymerase chain reaction and direct
sequencing 57 viral sequences from 47 individuals of North American,
Australian and Haitian origin infected with human immunodeficiency virus
type 1 (HIV-1), focussing on the V1 and V2 regions of gp120. There was
extensive length polymorphism in the V1 region, which rendered sequence
alignment difficult. The V2 hypervariable locus also displayed
considerable length variations, whereas flanking regions were relatively
conserved".(232) As far as Gallo is concerned, it is not even a
requirement that the "HIV" genome possess any genes whatsoever to be
pathogenic, "This suggests that defective virions such as RNA-free
particles and/or viral proteins expressed in the absence of particle
formation contribute to AIDS pathogenesis".(114)

8.1.4 In searching the HIV literature it is striking that to
date, not one single 9150 bp or any length of "full length HIV genome"
from fresh uncultured cells has been sequenced. "The low abundance of
HIV-1 proviral DNA in clinical samples is a barrier to full-genome
analysis of HIV-1 provirus as it occurs in vivo". All the "full-length
HIV genomes" sequenced so far have been from cultured cells in fact
"Completely sequenced full-length HIV-1 genomes in the current Los
Alamos data base have been derived, almost without exception, from HIV-1
isolates adopted to growth in continuous [leukaemic or transformed]
T-cell lines". As of late 1995 "only 19 sequences encompassing the
full-length, 10-Kb HIV-1 genome have been reported, and most derive from
HIV-1 isolates of genotype B expressed in continuous cell lines. Five of
the eight most prevalent genetic subtypes of HIV are without a single,
full-length, sequenced prototype".(193) At present it is also known
that: (a) patients belonging to the AIDS risk groups are exposed to high
doses of oxidising agents and that these agents have profound effects on
DNA and RNA; (74,79) (b) in cultures "HIV" cannot be detected unless
cultures are treated with chemical or physical oxidants including PHA;
(c) there are structural and functional abnormalities in the lymphocyte
genome of AIDS patients. "AIDS patients have shown increased levels of
spontaneous DNA repair synthesis (3 times higher), increased quantity of
single-stranded DNA breaks (11-18%), decreased ability to restore DNA
damage (2-2.5 times lower) compared to healthy persons";(233) (d)
according to Chermann and his colleagues, "Different populations of
distinct HIV-1 DNA fragments of highly variable size ranging from 600 bp
to full length provirus were present in PBMC from HIV-infected
persons...Defective genomes tended to gradually disappear after
activation of PBMC with phytohemaglutinin";(234) (e) According to the
HIV experts, the defective genomes are "rescued" by recombination and
this recombination is one of the main causes of "HIV DNA" complexity. If
this is the case one may ask: (i) can one exclude the possibility that
the 19 "full-length HIV genomes" described so far, even if they all had
the same length of 9150 bp and identical sequences are nothing more than
a chance finding among the many molecular species present in the
cultures, or even the uncultured lymphocytes, which have nothing to do
with a retroviral genome and which appeared as a result of either in
vivo or in vitro conditions or both and of natural selection?; (ii) if
there is such a high rate of recombination between the HIV genomes, is
it not possible that the same process takes place between the endogenous
retroviral genomes? If this is also the case, how does one know that the
19 "full-length HIV genomes" are nothing more than recombinations
between endogenous retroviral sequences, endogenous retroviral sequences
and cellular sequences, for example, non-retroviral retroelements? As
has been pointed out, HIV researchers seldom use controls and to date
those that have failed to use appropriate controls, that is, tissues or
cultures derived from similarly sick, non-AIDS individuals in which
experimental techniques and conditions employed are identical apart from
the presence of putative retroviral material. However, if HIV
researchers or others capable of mounting such experiments were
encouraged to put as much effort as they put into studying "HIV" from
lymphocytes of at risk patients into studying lymphocytes from patients
not at risk but: (a) who are exposed to agents (other than "HIV") and
doses similar to those in the high risk groups; (b) which have similar
structural and functional abnormalities as lymphocytes from AIDS
patients or those at risk; (c) using exactly the same methods and
culture conditions as those used by "HIV" researchers; can one exclude
the possibility that in another ten years time these researchers will
not be able to report "19 full-length HIV genomes" in these
individuals?

8.2 "For example, Jackson et al. have tested blood cells of 409
antibody-positives including 144 AIDS patients and 265 healthy people.
In addition 131 antibody-negatives were tested. HIV- specific DNA
subsets-defined in size and sequence by HIV-specific primers (start
signals for the selection amplification)-were found in 403 of the 409
antibody-positive, but in none of the 131 antibody negative people
(Jackson et al., 1990)".

8.2.1. Apparently, up until 1987 Jackson et al considered the
detection of RT (reverse transcription determined by transcription of
A(n).dT15 in cultures synonymous with HIV isolation! However, they had
an "isolation rate of 57% in patients with acquired immunodeficiency
syndrome". By 1988 the "reverse transcriptase assay was replaced with
the Abbot Laboratories HIV-1 antigen detection assay", which "primarily
detects the p24 core antigen of HIV-1...A culture was considered
positive for HIV-1 antigen if two serial supernatant samplings were
positive, with the later sampling showing greater activity"! "HIV-1 was
isolated from the PBMC of 141 (99.3%) of 142 HIV-1 antibody positive
patients".(235) In their 1990 paper Jackson et al reported that "Between
February 1987 and October 1988, peripheral blood mononuclear cells
(PBMC) from 409 individuals who were antibody positive for HIV-1 by
Western (immuno) blot (56 AIDS patients, 88 patients with ARC, and 265
asymptomatic individuals) were cultured". Using a sensitive technique
previously described", the p24 assay noted above, they reported that
"HIV-1 can be isolated from 100% (56 of 56) of AIDS patients, 99% (87 of
88) of ARC patients, and 98% (259 of 265) HIV- 1 antibody positive
asymptomatic individuals". Not one of "131 HIV- 1 antibody-negative
individuals has a positive culture". Using the same p24 assay (Abbot)
they tested the serum from 403 out of the 409 individuals. The test was
positive in 23/56 (42%) AIDS patients, 31/88 (57%) ARC patients and
44/259 (17%) asymptomatic antibody positive individuals. For unstated
reason(s) a positive serum test is considered proof for the detection of
"HIV-1 antigen in serum" while the same positive culture test is
considered proof for "HIV-1 isolation" from the culture. There are many
reasons to question the interpretation of the p24 assay: (a) The p24
assay is an antibody/antigen reaction and is subject to ubiquitous
background reactivity. In this context, even if "two serial supernatant
samplings with the later sampling showing greater reactivity", even if
double or triple, for example, 30 and 60 or 30 and 90, both readings may
be nothing else but background readings. Jackson and colleagues'
criteria are not even in agreement with those used by Ho et al which are
equally as arbitrary; "A culture was considered positive if the
concentration of p24 antigen in the supernatant exceeded 1000pg per
milliliter (typical cutoff value approximately 30pg per milliliter) on a
single determination or ò 200pg per milliliter on two or more
determinations".(51) In this regard it is important to note that no
amount of experimental variations and technological improvements in the
p24 test can,change the underlying nature of the test. The test solely
detects antibody/antigen reactivity and the reason underlying such
reactivity cannot be determined on the basis of an arbitrary cut off. A
priori, there is no reason why conditions leading to non-specific
reactivity should not be present at a sufficient level to drive the
reaction above cut off, nor any reason to prevent the reverse, that is,
specific reactivity below cut off. The only way to resolve this issue is
to compare reactivity with the presence or absence of HIV as determined
by virus isolation. To date, this has not been reported. Even without a
gold standard, the non-specificity of the p24 antigen test is so obvious
that it is accepted by no less an authority on HIV testing than Philip
Mortimer and his colleagues from the UK Public Health Laboratory
Service, "Experience has shown that neither HIV culture nor tests for
p24 antigen are of much value in diagnostic testing. They may be
insensitive and/or non-specific".(236) The fact that in experiments with
"serial dilution studies of culture supernatants" the p24 test is more
likely to be positive than RT is not proof that the p24 test is "at
least 100-fold more sensitive that reverse transcriptase assays".
Sensitivity for HIV can only be measured by the use of HIV isolation as
a gold standard;(237) (b) There are no scientific reasons and indeed no
commonsense reasons why reactions such as reverse transcription or
antibody/antigen reactions, even if specific for retroviruses, can be
considered proof for viral isolation. If these phenomena are considered
proof for virus isolation then both the pregnancy test, (measurement of
the protein áHCG in blood or urine using antibodies), or estimation of
cardiac enzymes in suspected myocardial infarction, must also be
considered proof for "isolation" of placenta or heart respectively.

8.2.2 To improve on the p24 assay, the DNA extracted from
frozen uncultured PBMC of their seven "antibody-positive culture
negative subjects" and "23 healthy heterosexual HIV-1 antibody negative,
culture negative individuals" was assayed by PCR. In addition, "In order
to compare the sensitivity and specificity" of the two tests, PCR and
culture, the PBMC of 59 seropositive and 20 seronegative individuals
were analysed by both tests. "Amplifications of HIV-1 were performed by
using a primer pair, SK38-39, which amplifies a 115-base-pair conserved
region of the gag gene (nucleotides 1551 to 1665 of HIV SF23: GenBank
accession no. K02007). The amplified product was detected by oligomer
hybridization, a technique in which a 32p-end-labeled probe (SK19) to
the nucleotide 1595 to 1635 gag region hybridizes in solution to one
strand of the amplified sequence. The probe-target duplex was then
resolved by electrophoresis on a 10% polyacrylamide gel and
autoradiographed". None of the seronegative individuals was reported to
have a positive PCR test. "All initial DNA samples from the 7 HIV-1
antibody-positive, culture-negative patients" were reported positive.
When the PCR and culture tests were compared, 57 of the 59 patients had
a positive PCR and 57 of the 59 patients had a positive culture. The 2
PCR negative individuals had positive cultures and the two culture
negative individuals had a positive PCR. The authors concluded, "We
isolated HIV-1 or detected HIV-1 DNA sequences from the PBMC of all 409
HIV-1 antibody-positive individuals. None of 131 HIV-1 antibody-negative
individuals were HIV-1 culture positive, nor were HIV-1 DNA sequences
detected by PCR in the blood specimens of 43 seronegative individuals.
In addition, HIV-1 PCR and HIV-1 culture were compared in testing the
PBMC of 59 HIV-1 antibody positive and 20 HIV-1 antibody negative
hemophiliacs. Both methods were found to have sensitivities and
specificities of at least 97 and 100% respectively...Our ability to
directly demonstrate HIV-1 infection in all HIV-1 antibody-positive
individuals provides definite support that HIV-1 antibody positivity is
associated with present HIV-1 infection".(52) In other words, Jackson et
al used the antibody tests as a gold standard for both the culture and
PCR tests and the PCR and culture tests as a gold standard for the
antibody test.

Jackson et al's claims are not even confirmed by other laboratories.
According to Jackson et al, up until 1990 only three small studies
reported "100% isolation rates of HIV-1 from AIDS patients". In all the
other studies, "HIV-1 was not isolated from 6 to 50% of HIV-1
seropositive AIDS cases reported. The culture recovery rate of HIV-1
from HIV-1 antibody positive asymptomatic patients has generally been
even lower, only 20 to 42% in some studies". The most recent situation
is best illustrated by a large WHO study published in 1994. Between
1992-93, 224 specimens were collected in Brazil, Rwanda, Thailand and
Uganda from asymptomatic "HIV positive" individuals. Serostatus was
first confirmed in the country of origin and then at the "centralized
laboratories responsible for confirming serology, virus isolation, virus
expression, and distribution of reagents (George-Speyer-Hans
Chemotherapentisches Forschunginstitut (GSH) in Frankfurt, Germany;
National Institute for Biological Standards and Control (NIBSC) in
London, United Kingdom,; and DAIDS/NIAID in Bethesda, Maryland, United
States". Using the method of Jackson et al, "of a total of 224 virus
cultures, 83 were positive (Isolation rate=37%)".(238) Jackson et al's
PCR results, like their culture results, are not reproducible in other
laboratories. For example, in the study conducted by Defer and her
colleagues, where the same samples were tested in "Seven French
laboratories with extensive experience in PCR detection of HIV DNA", the
data revealed that of 138 samples shown to contain "HIV DNA", 34 (25%)
did not contain "HIV antibodies" while of 262 specimens that did not
contain "HIV DNA", 17 (6%) did contain "HIV antibodies".197 In a paper
published in 1994 by researchers from The Laboratory of Molecular
Retrovirology Georgetown University, Chiron Corporation California,
Retrovirology Section, US National Institutes of Health, Maryland, the
authors noted that the PCR techniques are "exceedingly labor intensive
and suffer from laboratory-to-laboratory variation due to differences in
technique and operations" and that "in some reported studies there is no
correlation between p24 antigen levels and measurements of infectious
virions. Similarly, a decrease in p24 antigen level is not necessarily
associated with a positive clinical outcome". Because of this, to
"Monitor Human Immunodeficiency Virus Type 1 Burden in Human Plasma",
the authors used "the branched DNA signal amplification assay" which,
"offers improved sensitivity" and compared it with the "two other
standard assays for viral burden; end-point dilution plasma culture and
immune complex-dissociated (ICD) serum p24 antigen". They reported that
"HIV-1 DNA and ICD serum p24 antigen assays were done on serum samples
from 102 seropositive (Western blot-confirmed) patients who were being
screened for enrollment in clinical trials...of the 102 patients, 75
(74%) were positive for HIV RNA by the bDNA assay and 61 (60%) were
positive by the ICD p24 assay. Only a subset of patients (n=56: CD4 cell
range, 29-394; median 160) was tested for plasma viremia by viral
culture; 34 (61%) were culture-positive, while 50 (89%) were positive by
bDNA assay and 39 (70%) were positive by the ICD p24 assay".(239) How is
it then possible to claim that "virtually all people who contain HIV DNA
also contain antibodies against Montagnier's HIV strain" and "most, but
certainly not all people who lack HIV DNA contain no such
antibodies"?

CONCLUSION AND COMMENTS

Since Jackson et al did not test all 409 patients and all 131
antibody negative individuals for the presence of "HIV DNA" using PCR,
but tested only 66 patients and a maximum of 43 "antibody- negative"
individuals; did not sequence the amplified segments and did not
determine the specificity of the PCR by using the only valid gold
standard, HIV isolation, it was not possible for them to report "HIV
specific DNA subsets...in 403 of the 409 antibody- positive, but none of
the 131 antibody-negative people". Furthermore, Jackson et al
acknowledged that their PCR method did not prove the existence of the
full-length HIV genome but only "that AIDS patients as well as HIV-1
antibody-positive asymptomatic individuals harbor HIV-1 genetic
material". In addition, for their PCR determinations, Jackson et al used
a small fragment of the gag gene as a primer. But: (a) since the best
known HIV experts agree that the gag genes of retroviruses are
homologous, Jackson et al's negative PCR results in all 43
"antibody-negative" individuals who must at least have had the
retrovirus present "in all of us", remains unexplained; (b) finding a
positive PCR result using a small fragment of the gag gene as primer is
not proof for the existence of the "full-length HIV genome" or even for
the existence of the "full-length HIV gag gene". As has been already
mentioned, by 1989 researchers at the Pasteur Institute concluded that
"the task of defining HIV infection in molecular terms will be
difficult". In fact, as far back as 1973, retrovirologists were aware
that the unusual nature of retroviruses "will prove a stumbling block to
any genetic analysis of RNA tumor viruses".(240) Yet, at least some HIV
experts, including Jackson et al insist on defining HIV infection in
genetic terms. On the other hand, an analysis of the presently available
data on retroviruses shows that all retrovirologists seem to agree that
the single most decisive factor in proving the existence of a unique
retrovirus is the existence of specific antibodies, its importance well
illustrated by the history of the discovery and subsequent demise of
HL23V (see 5.4). As far as HIV is concerned, it is well known that the
only evidence considered to prove the HIV theory of AIDS is a
correlation between the clinical syndrome and a positive antibody test.
Less well known is the fact that in the four papers published in Science
in May 1984, Gallo and his colleagues claimed that in contradistinction
to Montagnier and his colleagues, he and his colleagues achieved "true
isolation". However, it is of pivotal significance that the only
difference between the experiments performed by the two groups is that
Gallo's group employed a leukaemic cell line from which they were able
to obtain abundant "HIV antigens" and thus could perform significantly
more antibody tests. Given the crucial status retrovirologists accord to
specific antibodies proving the existence of a unique retrovirus, and
its role in pathogenicity, proof of antibody specificity would appear to
be mandatory. The specificity of the HIV antibody tests can be
determined only by the use of HIV isolation as a gold standard. To date
this has not been done and at present would seem impossible because
nobody has fulfilled even the first step in the only scientifically
valid method for retroviral isolation, that is, electron microscopic
demonstration of particles with the morphological characteristics of
retroviruses banding in sucrose density gradients at the density of 1.16
gm/ml. In addition, "HIV" can only be "isolated" from a minority of
individuals who has a positive antibody test.

Furthermore, as in the case of HL23V, there is evidence that
antibodies present in human sera which react with "HIV proteins" are
also non-specific: (a) "One half of the molecular weight of gp120 is
represented by oligomannosidic oligosaccharides...Polyclonal antibodies
to mannan from yeast also recognize the carbohydrate structure of gp120
of the AIDS virus";(241) (b) "The immunochemical determinants of the
antigenic factors of Candida albicans display a high identity with the
glycoprotein (gp) 120 of HIV-1: they contain à(12) and à(13) linked
mannose terminal residues";(242) (c) antibodies to the mannans of
Candida albicans "block infection of H9 cells by HIV-1" as well as the
binding of lectins to gp120;(242) (d) recognition of gp120 by antibodies
to a synthetic peptide of the same antigen was "partially abolished if
it was absorbed with the total polysaccharide fraction of C. albicans"
while the antigen recognition by antibodies to "gp120 from human T cell
lymphotropic virus type IIIB", "was totally blocked". From these data
the authors concluded: "These results indicate that mannan residues of
C. albicans can serve as antigens to raise neutralizing antibodies
against HIV infection;(242) (e) "normal human serum contains antibodies
capable of recognizing the carbohydrate moiety of HIV envelope
glycoproteins...from 100ml of human serum approximately 200ug of MBIgG
was recovered [MBIgG=mannan-binding IgG]...MBIgG bound to HIV envelope
glycoproteins gp160, gp120 and gp41";(243) (f) researchers from the
University of Rome infected healthy mice with an E. coli
lipopolysaccharide (LPS) and reacted their sera with two synthetic
peptides, one encompassing gp120 V3 loop of "HIV-1 MN" and the other
"representing a gp41 immunodominant epitope". The "LPS-treated mice
showed a significant antibody reactivity" with the two peptides. (V
Colizzi et al., personal communication). (g) Kashala, Essex and their
colleagues have shown that antibodies to carbohydrate containing
antigens such as lipoarabinomannan and phenolic glycolipid that
constitute the cell wall of Mycobacterium leprae, a bacterium which
"shares several antigenic determinants with other mycobacterial species"
cause "significant cross- reactivities with HIV-1 pol and gag proteins".
This led the authors to warn that among leprosy patients and their
contacts there is a "very high rate of HIV-1 false-positive ELISA and WB
results", that "ELISA and WB results should be interpreted with caution
when screening individuals infected with M. tuberculosis or other
mycobacterial species", and furthermore that "ELISA and WB may not be
sufficient for HIV diagnosis in AIDS-endemic areas of Central Africa
where the prevalence of mycobacterial diseases is quite high".(244)

Not only mycobacteria (M. leprae, M. tuberculosis, M. avium-
intracellulare) but also the walls of all fungi (Candida albicans,
Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum
including Pneumocystis carinni),(245-247) contain carbohydrate
(mannans). One hundred per cent of AIDS patients (even those with "No
candida clinically") have Candida albicans antibodies leading
researchers from St. Bartholomews and St. Stephen's Hospitals to state:
"It is possible that candida may act as a cofactor in the development of
overt AIDS in HIV infected individuals".(248) It may also be of interest
to note that in gay men the only sexual act which is a risk factor for
seroconversion is passive anal intercourse (exposure to semen) (249)
that mannose is present in both sperm and seminal plasma.(250) Since
antibodies to mannans react with the "HIV proteins" then, as Essex and
his colleagues have pointed out for mycobacterial infection in Africa,
one would expect the sera of all people infected with fungi and
mycobacteria to cross-react with the "HIV-1 glycoproteins" as well as to
cause "significant cross-reactivities with HIV-1 pol and gag proteins".
Given the fact that individuals with fungal and mycobacterial infections
have antibodies which may produce a positive "HIV" antibody test even in
the absence of "HIV", how can one assert that: (a) PCP, candidiasis,
cryptococcosis, coccidioidomycosis, histoplamosis, tuberculosis or
Mycobacterium avium-intracellulare disease, that is, the vast majority
of the opportunistic infections (88% of AIDS cases diagnosed between
1988 and 1992 had one or more fungal or mycobacterial infections251)
which signify AIDS are caused by HIV on the basis of a positive antibody
test? (b) that a positive antibody test in individuals with fungal and
mycobacterial infections proves HIV infection?

Indeed, as in the case of HL23V, is it only a matter of time before
HIV researchers accept that there may be no such entities as specific
HIV antibodies? As a consequence, will the compilation of phenomena
inferred as proof of the existence of the human immunodeficiency virus,
pass into history as "non-viral material altogether"? *

30. Chamaret S, Squinazi F, Courtois Y, et al. Presence
of anti-HIV antibodies in used syringes left out in public places,
beaches or collected through exchange programs. XIth International
Conference on AIDS 1996, Vancouver.

38. Barbacid M, Bolognesi D, Aaronson SA. Humans have
antibodies capable of recognizing oncoviral glycoproteins: Demonstration
that these antibodies are formed in response to cellular modification of
glycoproteins rather than as consequence of exposure to virus. Proc Natl
Acad Sci U S A 1980;77:1617-1621.

69. Small JV, Langanger G. Organisation of actin in the
leading edge of cultured cells: influence of osmium tetroxide and
dehydration on the ultrastructure of actin meshworks. J Cell Biol
1981;91:695-705.

207. Mayers MM, Davenny K, Schoenbaum EE, et al. A
prospective study of infants of human immunodeficiency virus
seropositive and seronegative women with a history of intravenous drug
use or of intravenous drug-using sex partners, in the Bronx, New York
City. Pediatrics 1991;88:1248-1256.